- Volume 139, Issue 4, 1993

Sexual adhesion in the green alga Chlamydomonas eugametos is mediated by hydroxyproline-containing glycoproteins (agglutinins). The agglutinins were metabolically labelled with [35S]sulphate. The sulphur-containing amino acids methionine and cysteine were not detectably labelled; little or no sulphated tyrosine was detected. This indicated that the radioactive label had been largely or exclusively incorporated into sulphated sugar residues. Acid hydrolysis released essentially all radioactivity as free sulphate. Short periods of hydrolysis released sulphated oligosaccharides. β-Elimination released about 20–25% of the sulphated saccharides, indicating that these are O-linked to serine or threonine or both. The remaining sulphated sugar chains are presumably O-linked to hydroxyproline. It is proposed that the sulphated sugars of the agglutinins play a role in sexual adhesion.

The sensitivity of elongation factor Tu (EF-Tu) from different species of bacteria to the EF-Tu-binding antibiotics efrotomycin, pulvomycin and MDL 62879 was tested by measuring the effect of these antibiotics on cell-free protein synthesis systems. EF-Tu from four different Gram-negative species was sensitive to all three antibiotics. Among Gram-positive bacteria, EF-Tu of Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis was resistant to efrotomycin and less sensitive to pulvomycin than EF-Tu of Gram-negative bacteria. EF-Tus from streptococci were significantly less sensitive than EF-Tus from Gram-negative bacteria to both efrotomycin and pulvomycin. All of the EF-Tus were sensitive to MDL 62879. The same sensitivity pattern emerged from GDP exchange assays, performed with partially purified EF-Tu from different bacterial species and pure Escherichia coli EF-Ts. These results suggest that the site of action of MDL 62879 is more conserved among bacterial species than those of efrotomycin and pulvomycin. Heterogeneity of EF-Tus from different bacterial species was also reflected in differences in their apparent molecular masses estimated by SDS-PAGE. EF-Tus from the Gram-positive species had higher molecular masses than those from all but one of the Gram-negative species.

Nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Bacillus cereus DSM 31 was enriched 260-fold. The molecular mass was determined by gel filtration to be 270 kDa (± 25 kDa). The enzyme was highly specific for the coenzyme NAD(H) and catalysed both the formation and the oxidation of glutamate. Apparent K m values of 7.7 mM for glutamate and 0.56 mM for NAD+ during oxidative deamination were measured. Both in crude cell-free extracts and in enriched preparations the enzyme was extremely unstable, especially at low temperatures. The loss of activity in the cold was found to be due to the dissociation of the holoenzyme into catalytically inactive subunits of molecular mass 48 kDa (±5 kDa), indicating that the native enzyme has a hexameric structure. The activity was restored under certain conditions, and no instability of the enzyme in the cold was observed in undisrupted cells.

Alkaline isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) activity was detected in the culture medium of an alkalophilic strain of Bacillus sp., designated KSM-3309, which was isolated from a soil sample. This novel enzyme was purified to homogeneity from the culture filtrate by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and DEAE-Bio-Gel A, and gel filtration on Sephacryl S-200. The purified enzyme had a pH optimum of approximately 9·0, and displayed maximum catalytic activity at 55 °C. The enzyme had a molecular mass of 65 kDa, as determined by both SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200. The isoelectric point was 4·2. This enzyme cleaved the branching points of both amylopectin and glycogen, and incubation of the enzyme with these glucans caused large increases in coloration of the iodine reagent. Amylose, pullulan and maltose were practically insensitive to the enzyme. The enzyme activity was inhibited by Hg2+ ions and by N-bromosuccinimide, but the thiol inhibitors iodoacetate, 4-chloromercuribenzoate and N-ethylmaleimide had either no effect or a slightly inhibitory effect. β-Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.

The DNA base sequence of the Pseudomonas sp. NK87 gene (P-nylB) for 6-aminohexanoate-dimer hydrolase (P-EII), a xenobiotic-compound-degrading enzyme, was determined. It has an open reading frame of 1188 bp, initiated by ATG and terminated by TAG, and coding for 396 amino acids. The base sequence of the open reading frame has 53% sequence similarity to that of the gene for the same enzyme of Flavobacterium sp. KI72 (F-nylB) and 35% sequence similarity with respect to the deduced amino acid sequence. The P-EII enzyme was purified from an Escherichia coli clone in which the P-EII gene was highly expressed. The P-EII enzyme was inhibited by a serine protease inhibitor, diisopropyl fluorophosphate, as was the F-EII enzyme. Double reciprocal plots obtained from various concentrations of 6-aminohexanoate-dimer indicated that the k cat value of the P-EII enzyme (9.2 s−1) was approximately half that of the F-EII enzyme (19 s−1), and the P-EII enzyme had higher affinity toward this substrate (K m for P-EII, 0·6 mm; K m for F-EII, 15 mm). The P-EII enzyme had a temperature optimum of 48 °C, and a pH optimum of 7·5. It is speculated that since the P-nylB and F-nylB genes are more diverged from each other than the corresponding nylA genes, the latter may have evolved more recently.

Pseudomonas putida ATCC 17453 grew with either (+)- or (−)-camphor as sole carbon source. Enantiomer-specific ‘biological Baeyer-Villiger’ monooxygenases were synthesized irrespective of the camphor isomer used for growth. The two enzymes are probably the products of separate genes but showed many similarities. Each consisted of two electrophoretically indentical subunits, bound flavin mononucleotide (FMN) non-covalently and accepted electrons from an induced NADH dehydrogenase which interacted with the FMN bound to the oxygenating component. They showed minor differences in M r with 3,6-diketocamphane 1,6-monooxygenase being the smaller enzyme. Isoelectric focussing showed the two enzymes to have different acidic pI values. Polyclonal antibodies raised against 3,6-diketocamphane 1,6-monooxygenase also cross-reacted with 2,5-diketocamphane 1,2-monooxygenase and its subunits.

The structure of the surface of colonies of various Gram-negative and Gram-positive bacteria was examined by transmission electron microscopy. The results indicate that bacterial colonies in the course of their development produce a film which becomes thicker with increased duration of growth. The basic part of the film is an elementary membrane, which is a stable structure with a large surface area. The inner and outer surfaces of the film membrane are covered by amorphous layers. These layers are thicker in the surface film of Gram-negative bacterial colonies than in those of Gram-positive bacteria. Membrane vesicles from the bacterial colonies take part in the formation of the surface film. The presence of the film on the surface of the colonies of different bacteria suggests that this structure may play an important role.

Rhapidosomes are tubular microstructures composed of proteins that are found in a variety of bacteria and algae. These structures, which are resistant to disruption by many denaturing agents, have potential application as a biomaterial and may serve as a new model for the study of self assembly. When rhapidosomes were purified and analysed by SDS-PAGE the presence of three proteins with molecular masses of 53, 29 and 18 kDa was revealed. However, lysozyme treatment of the rhapidosome preparations containing the three proteins resulted in the selective release of the 18 kDa protein from the rhapidosome complex. N-Terminal sequence analysis and amino acid composition analysis were performed on all three proteins. The amino acid composition of the 18 kDa protein closely matched the amino acid composition of protein H (a peptidoglycan-associated protein from Pseudomonas aeruginosa). These results suggest that the 18 kDa protein may be a contaminating peptidoglycan-associated protein and not part of the rhapidosome structure. Western blot analysis using antisera raised against rhapidosomes revealed that the 53 kDa component protein does not react with the antisera. We speculate that the 53 kDa protein may form the inner core of the rhapidosomes, whilst the 29 kDa protein forms the outer sheath of these structures.

In a screening programme, many new compounds were found that induced myxospore formation in Stigmatella aurantiaca strain Sg a1. The most efficient compounds were indole and indole derivatives, e.g. 3-methylindole. With an inducing concentration of 0·07 mm, this compound was about 2000 times more efficient than glycerol. Indole compounds, which have also been found as natural metabolites of myxobacteria, are thus candidates for auto-inducers within the fruiting bodies. Mutants were isolated that were resistant to particular inducers. They often showed cross-resistance to many other inducers. By the sporulation response of three different mutants, the inducers could be classified into four groups. Strains resistant to inducers of, e.g. group I were still sensitive to inducers of groups II and III, and vice versa. This classification was supported by newly discovered inhibitors of induced sporulation. These inhibitors, oxindole and pyrrole, were specific for inducers of group I. The grouping is further supported by differences in the kinetics of myxospore formation. We postulate that S. aurantiaca has three inducer-specific, independent receptors.

A study was made of the antagonistic effect of Ruminococcus flavefaciens on the cellulolytic activity of Neocallimastix frontalis. An extracellular factor inhibiting the cellulolytic activity of the fungus was detected in the bacterial supernatant. The antagonistic factor, which precipitated with ammonium sulphate at 40% saturation, was temperature-sensitive and was destroyed at temperatures above 60 °C. After separation by anion-exchange chromatography, sequential precipitation, dialysis and SDS-PAGE, two protein species of 100 and 24 kDa were identified as being involved in this antagonistic effect. It is not known whether the proteins are two subunits of a single protein or represent two different proteins. The inhibitory factor, which is not a bacterial cellulase, did not affect fungal growth, but it inhibited the activity of the fungal cellulases.

The indigenous plasmid pIJ101 is the parent of many cloning vectors used in Streptomyces. One early pIJ101 derivative, pIJ702, has been particularly widely used. pIJ702 lacks sti:cop/korB and accumulates single-stranded DNA (ssDNA). The 1.2 kb BclI-BclI sti:cop/korB and 0.7 kb SpeI-BclI sti regions were isolated from pIJ101 and cloned into pIJ702 at the PstI site in both orientations. No ssDNA was detected in constructs containing sti present in its correct orientation with respect to the basic replicon, with or without cop/korB. Constructs which contained sti in the reverse orientation did accumulate ssDNA. Thus, sti is only active as the site for second-strand synthesis in its natural orientation. Furthermore, sti inserted in either orientation into the structurally unstable pIJ702-pUC8 shuttle vectors prevented them from rearranging in S. lividans. The sti function was defined to a 0.53 kb SpeI-SacII fragment and the probable site for second-strand initiation (ssi) was identified.

One of the chitinases of Streptomyces lividans, chitinase C, was encoded by a 2 kb smaI-XhoI restriction fragment contained in the recombinant plasmid pEMJ7. DNA sequence analysis of this region revealed the presence of two open reading frames (ORF1 and ORF2) which had opposite orientations. Northern analysis showed that only the mRNA complementary to ORF1 was transcribed, and that this transcription was induced by chitin and repressed by glucose. ORF1 showed a codon distribution typical of Streptomyces. A sequence identical to that of the N-terminus of mature secreted chitinase C was found from amino acid residue 31 in the deduced amino acid sequence of ORF1 (619 amino acids), implying that ORF1 encodes a pre-protein of chitinase C. The pre-protein of chitinase C consisted of four discrete domains. The 30 amino acid N-terminal sequence, domain 1, was characteristic of a signal peptide. Domain 2 consisted of 105 N-terminal amino acids of mature chitinase C, and was similar to cellulose-binding domains of several cellulases. Domain 3 (94 amino acids) showed homology with type III homology units of fibronectin. Domain 4, a C-terminal 390 amino acid sequence, is probably the catalytic domain of the chitinase, since it exhibited identity with several other chitinolytic enzymes.

The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.

The integrating plasmid pCOA18, comprising pUC18 linked to a mutated coagulase (coa) gene from Staphylococcus aureus, and constructed by substituting coa sequences with a tetracycline (Tc)-resistance marker (∆coa::Tcr), was transformed into Staphylococcus aureus RN4220, where it underwent recombination with the chromosomal coa locus. Allele-replacement mutants were recovered at a low frequency directly after transformation. The majority of transformants carried pCOA18 integrated in the chromosome by a single Campbell-type recombination event. The majority of integrants contained tandem repeats of pCOA18 and expressed high levels of resistance to Tc (> 30 μg ml−1) compared to the single-copy integrants and allele-replacement mutants (15 μg ml−1). Transduction of a single-copy integrant to a Coa+ recipient allowed the cointegrant to be resolved and allele-replacement recombinants to be selected. In addition, growth of a single-copy integrant on high concentrations of Tc (> 30 μg ml−1) selected for amplified derivatives at a frequency of 10−5. It was estimated that up to 19 copies of pCOA18 could occur in a tandem array in the chromosome.

The rpmB,G operon of Escherichia coli codes for the synthesis of ribosomal proteins L28 and L33. In one mutant strain (TP28), these two proteins are made at about half their normal rates, ribosome assembly is greatly perturbed and precursor particles accumulate. The mutation in strain TP28 is in a Shine-Dalgarno sequence in the leader region of the rpmB,G messenger RNA. Another mutant, strain AM108, makes neither protein because it has the mobile element IS1 inserted into the rpmB coding sequence. Surprisingly, ribosome assembly in this strain is virtually normal with respect to growth rate. Strain AM90, which fails to make protein L33, has the element IS3 inserted into rpmG and also shows no major defects in ribosome assembly.

The gene (nir) encoding the copper-containing nitrite reductase (NIR) of a denitrifying bacterium, Alcaligenes faecalis S-6, was cloned by a synthetic oligonucleotide-probing method. The nucleotide sequence of the cloned DNA fragment revealed the primary structure of the NIR precursor containing the N-terminal signal sequence for secretion. A nucleotide sequence, possibly recognized by a transcriptional regulator resembling FNR was found upstream of the structural gene. When the cloned gene was expressed in Escherichia coli under the control of the lac promoter at 37 °C, NIR was produced as large inclusion bodies and little activity was detected. When cultivation was at 20 °C, most of the NIR was detected in the soluble fraction and a significant portion of the protein was translocated into the periplasmic space, accompanied by removal of its signal sequence.