- Volume 137, Issue 2, 1991

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum β-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum β-lactam antibiotics and with constitutively overproduced chromosomal β-lactamase in the periplasm. The binding of structurally different β-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1·5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested β-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced β-lactamase did not prevent PBP-β-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific β-lactams.

The production of two haemolysins, thermostable direct haemolysin (Vp-TDH) and a Vp-TDH-related haemolysin (Vp-TRH), by clinical isolates of Vibrio parahaemolyticus has previously been reported. Here we describe a third type of haemolysin (named Vp-TDH/I), which is produced by a clinical isolate (strain TH012) that is Kanagawa phenomenon negative. Vp-TDH/I was purified by a series of column chromatographies on DEAE-Sephadex A25, hydroxyapatite, Sepharose 4B and Mono Q. By physicochemical, biological and immunological analyses, Vp-TDH/I was demonstrated to be similar, but not identical, to Vp-TDH and Vp-TRH. The gene encoding Vp-TDH/I was cloned and the deduced amino acid sequence of Vp-TDH/I confirmed that Vp-TDH/I has a sequence different from those of previously known Vp-TDH and Vp-TRH. Not only purified Vp-TDH/I but also live cells of the Vp-TDH/I-producing strain induced fluid accumulation in ligated rabbit intestine. We conclude that this clinical isolate produces a new type of Vp-TDH-related haemolysin, which may be involved in the pathogenesis of this organism.

Growth of Fusarium oxysporum on heat-killed Bacillus subtilis cells was accompanied by the loss of bacterial cytoplasmic contents, and this ‘cytolysis’ could be catalysed in heat-treated bacteria by the fungal culture fluids. In electron micrographs the bacterial walls appeared undamaged, and the absence of wall-lytic enzymes was confirmed by use of isolated bacterial walls as substrate. Appearance of cytolytic activity in cultures was paralleled by the production of proteolytic activity in the cultures. Proteolysis and cytolysis had similar pH optima at 8·8–9·0. Cultures grown on casein, but not glucose, produced high cytolytic activity. Rapid cytolysis occurred when heat-treated B. subtilis cells were incubated with trypsin, subtilisin or pronase E. Viable bacteria, however, were not attacked, either by concentrated culture fluids or by the commercial protease preparations.

A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers’ yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4·5-fold), and increased TPS activity at 5 mm-UDP-glucose/10 mm-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (K i about 5 mm), but increased TPP activity about six-fold (K a about 4 mm). Phosphate (6 mm) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.

This paper describes periplasmic c-type cytochromes from two strains of Paracoccus denitrificans NCIB 8944 grown in heterotrophic or methylotrophic conditions. It is shown that the functions of two monomeric, monohaem cytochromes induced during growth on methanol have been wrongly designated in previous work. The CO-reactive cytochrome c 553 (30 kDa) is not the electron acceptor for methanol dehydrogenase; this is shown to be the role of the cytochrome c 552 (22 kDa). The monomeric 45 kDa cytochrome induced in conditions of oxygen insufficiency is a dihaem c-type cytochrome and does not contain haem b as previously assumed. In addition to these cytochromes, the Oxford strain of NCIB 8944 contains two cytochrome c complexes. One of these (150 kDa), produced in relatively small amounts, consists of a non-haem protein plus four haemoproteins (28, 33, 41 and 47 kDa). The second complex is a novel dimeric multi-haem cytochrome c (46 kDa) which constitutes about 25% of the periplasmic c-type cytochrome. It reacts with CO and has no methionine ligands. One subunit (16 kDa) has two low-spin haems; the larger subunit (30 kDa) has three haems which have low-spin characteristics in the oxidized state and are high-spin in the reduced state. The subunits were readily separated at pH 12 and could be subsequently reconstituted into a complex indistinguishable from the original. The 30 kDa subunit was denatured on prolonged exposure to high pH, which also converted it to a low-spin cytochrome. No function could be designated for these novel c-type cytochrome complexes.

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

The coding characteristics of four plasmids expressing a protein (BCP) which comigrates with bacterioferritin were examined and the nucleotide sequence of a common 1985 bp segment from the 53 min region of the Escherkhia coli linkage map was determined. Three open reading-frames (orf1, orf2 and orf3) were detected, and orf2 (bcp, 156 amino acid codons) appeared to encode the bacterioferritin comigratory protein, BCP. The translation product of orf3 (205 amino acid codons) resembled the iron-sulphur protein component (DMS B subunit) of the anaerobic dimethylsulphoxide reductase complex of E. coli.

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7·2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the β-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32·5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid squence were identified when the β-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.

Myxococcus xanthus mutants defective in myxospore germination have been isolated both by a selective and by a non-selective method after UV or Tn5-lac-induced mutagenesis. The ability of these mutants to germinate in germinant solutions other than those used for their isolation has been tested. Six of seven mutants isolated behaved as germination-defective in all germinants. Germination of the seventh mutant was conditional on the germinant used, being normal in Casamino acids but defective in a Casitone-based medium. Genetic analysis of the four mutant strains carrying Tn5-lac insertions revealed that the transposon had disrupted a different locus in each mutant, so that the four mutants defined four unlinked loci involved in the germination process (gerA, gerB, gerC, gerD). Strain MR307 was studied in more detail. Cloning of the gene affected in this mutant, gerC, and construction of merodiploids revealed that the wild-type allele is dominant over the mutated one. In vitro construction of lacZ fusions allowed study of gerC expression throughout the M. xanthus life cycle, revealing that the gene affected by insertion at ΩMR307 is developmentally regulated.

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.

General properties of S-adenosylmethionine decarboxylase (SAMDC) from Mucor rouxii were studied. Dormant spores of the fungus did not contain detectable levels of the enzyme, but it started to be synthesized at early stages of spore germination. Kinetics of synthesis changed before emergence of the germ tube, with a corresponding increase in a second SAMDC activity which, in contrast to the one originally synthesized, was not activated by putrescine. Development of the second enzyme activity required de novo protein synthesis. Neither enzymic activity was stimulated by Mg2+. Addition of the SAMDC inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG) stopped fungal development in growth phase Ia: cells became spherical and showed ultrastructural alterations. Although MGBG inhibited polyamine formation, it barely inhibited protein and RNA biosynthesis during the first hour of incubation. However, at later periods, biosynthesis of both macromolecules was strongly decreased. When MGBG was added to growth media 3 h after inoculation of spores, it did not affect spore germination and outgrowth. A hypothesis for two different roles of spermidine and putrescine in spore germination is discussed.

Aspergillus nidulans produces an extracellular β-d-fructofuranoside fructohydrolase (invertase) when grown on a medium containing the β-fructofuranosides sucrose or raffinose, indicating that synthesis is subject to induction by the substrate. On a growth medium containing sucrose, production was maximal at 15 h in cultures incubated at 28 C°. After this time the level of detectable invertase in the cultures declined. A proportion of the enzyme was secreted during the linear growth phase of the fungus. Various sugars were investigated for induction of invertase, but only the two β-fructofuranosides induced high production levels; with the other sugars, the enzyme was produced only at a low constitutive level. Mycelium grown under repressive conditions (1% glucose), rapidly produced invertase when transferred to sucrose-containing medium. After 80 min the invertase level in these cultures was 26-fold higher than the constitutive level. The repressive effect of other sugars, e.g. glucose and xylose, on invertase production was also demonstrated in this experimental system.