- Volume 142, Issue 12, 1996

Haemophilus influenzae acquires iron from the iron-transporting glycoprotein transferrin via a receptor-mediated process. This involves two outer-membrane transferrin-binding proteins (Tbps) termed Tbp1 and Tbp2 which show considerable preference for the human form of transferrin. Since the Tbps are attracting considerable attention as potential vaccine components, we used transferrin affinity chromatography to examine their conservation amongst 28 H. influenzae type b strains belonging to different outer-membrane-protein subtypes as well as six non-typable strains. Whole cells of all type b and non-typable strains examined bound human transferrin; whilst most strains possessed a Tbp1 of approximately 105 kDa, the molecular mass of Tbp2 varied from 79 to 94 kDa. Antisera raised against affinity-purified native H. influenzae Tbp1/Tbp2 receptor complex cross-reacted on Western blots with the respective Tbps of all the Haemophilus strains examined. When used to probe Neisseria meningitidis Tbps, sera from each of four mice immunized with the Haemophilus Tbp1/2 complex recognized the 68 kDa Tbp2 of N. meningitidis strain B16B6 but not the 78 kDa Tbp2 of N. meningitidis strain 70942. Serum from one mouse also reacted weakly with Tbp1 of strain B16B6. Apart from a weak reaction with the Tbp2 of a serotype 5 strain, this mouse antiserum failed to recognize the Tbps of the porcine pathogen A. pleuropneumoniae. However, a monospecific polyclonal antiserum raised against the denatured Tbp2 of Neisseria meningitidis B16B6 recognized the Tbps of all Haemophilus and Actinobacillus strains examined. Since H. influenzae forms part of the natural flora of the upper respiratory tract, human sera were screened for the presence of antibodies to the Tbps. Sera from healthy adults contained antibodies which recognized both Tbp1 and Tbp2 from H. influenzae but not N. meningitidis. Convalescent sera from meningococcal meningitis patients contained antibodies which, on Western blots, recognized the Tbps2s of both pathogens. These data demonstrate the existence of shared epitopes on the Tbps of H. influenze, N. meningitidis and A. pleuropneumoniae despite their transferrin species specificity.

Summary: With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II - membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the mycoides cluster or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the mycoides cluster.

Growth of the thermoacidophilic archaeon Sulfolobus shibatae was tested on a range of carbon and nitrogen sources. Optimal defined and complex growth media were developed and growth conditions in both shake flask and fermenter cultures were optimized. Better growth was observed on maltose in particular and disaccharides in general than on monosaccharides. Moreover, maltose utilization was not repressed in the presence of glucose which suggests that glucose is not the preferred substrate of S. shibatae. Uptake studies putatively identified two saturable, constitutive maltose transport systems, a high-affinity, possible membrane-binding system with a K m of 20 μM and a V max of 218 nmol min−1 (mg protein)−1, and a low-affinity, proton-dependent system with a K m of 158 μM and a V max of 680 nmol min−1(mg protein)−1. Both systems showed differential responses to treatment with 2,4-dinitrophenol and arsenate, and differed from other maltose transport mechanisms described in being constitutive under all conditions tested and not repressed by glucose.

Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa 3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,NȲ,NȲ-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate-and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc 1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc 1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c 1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc 1 complex. Haem staining revealed that haem proteins of M r 31000 and M r 23000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M r 23000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c 1 and, in its absence, the protein of M r 23000 does not remain associated with the membrane. Formation of this M r 23000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc 1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.

Protamine is a polycationic peptide found in the nuclei of sperm of different animal species. While it has long been known to have antimicrobial properties, its mode of action has remained elusive. We have investigated the mechanism of action of protamine and established that this peptide exerts its antibacterial effect without causing cell lysis or permeabilization of the cytoplasmic membrane. Respiring cells were more susceptible than nonrespiring cells, and loss of viability could be prevented by incubation at low pH or the addition of respiratory poisons. This indicates that protamine activity is influenced by the electrical membrane potential (ΔΨ): increased killing occurs at higher ΔΨ values. Protamine caused inhibition of proline uptake, rapid efflux of proline from preloaded cells, and a reduction in the cellular ATP content. Furthermore, protamine-treated cells first lost the ability to accumulate leucine and then could not carry out protein synthesis. Cumulatively, our data indicate that protamine disrupts energy transduction and nutrient uptake functions, and suggest that the cytoplasmic membrane is the target of protamine action.

In purple sulphur bacteria of the family Chromatiaceae sulphite oxidation via intermediary formation of adenylylsulphate is an enzymologically well characterized process. In contrast, the role of an alternative direct oxidation pathway via the enzyme sulphite: acceptor oxidoreductase has not been resolved. This paper reports the cloning of the genes encoding the adenylylsulphate-forming enzyme adenosine-5′-phosphosulphate (APS) reductase from Chromatium vinosum strain D (DSM 180T), a representative of the purple sulphur bacteria, and the construction of mutations in these genes by insertion of a kanamycin Ω cartridge. The mutated genes were transferred to C. vinosum on suicide vectors of the pSUP series by conjugation and delivered to the chromosome by double homologous recombination. Southern hybridization and PCR analyses of the recombinants obtained verified the first insertional gene inactivation in purple sulphur bacteria. Enzymological studies demonstrated the absence of APS reductase from the mutants. Further phenotypic characterization showed no significant effect of APS reductase deficiency on the sulphite-oxidizing ability of the cells under photolithoautotrophic growth conditions. In the wild-type as well as in mutant strains, tungstate, the specific antagonist of molybdate, led to the intermediary accumulation of sulphite in the medium during sulphide oxidation and strongly inhibited growth with sulphite as photosynthetic electron donor; this indicates that a molybdoenzyme, probably sulphite: acceptor oxidoreductase, is the main sulphite-oxidizing enzyme in C. vinosum. Specific inactivation of selected genes as developed for C. vinosum in this study provides a powerful genetic tool for further analysis of sulphur metabolism and other metabolic pathways in phototrophic sulphur bacteria.

Summary: Aminoglycoside antibiotics, used to treat bacterial infections by interfering with proofreading during protein synthesis, cause sensorineural hearing loss in genetically susceptible individuals. The only aminoglycoside-hypersensitivity mutations which have been described in humans are in the mitochondrial 12S rRNA gene, potentially allowing increased antibiotic binding to mitochondrial ribosomes. To identify additional predisposing mutations, a yeast model system was used to isolate genes which interact with or bypass the effects of aminoglycoside antibiotics. A novel yeast gene was isolated which, in high copy, confers neomycin resistance to yeast transformants. The neomycin-resistance 1 gene (NEO1) encodes a potential 1151 aa integral membrane protein, most homologous to the yeast DRS2 gene product, a Ca2+-ATPase involved in cytoplasmic ribosome assembly. The N-terminus of Neo1p is partially homologous to abrin A-chain, another protein which interacts with cytoplasmic ribosomes. Mutagenesis experiments demonstrate that the NEO1 product is essential for vegetative growth and that the drug-resistance phenotype requires ATPase function.

Summary: The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as a rho-independent transcription terminator sequence for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outer-membrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (< 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.

Summary: The nucleotide sequence of wprA, a protease-encoding gene of Bacillus subtilis 168, is reported. The gene, expressed during the exponential growth phase, belongs to a monocistronic operon. WprA is a 96 kDa polypeptide endowed with a signal peptide, as well as a propeptide. Upon processing and export, it gives rise to two previously identified cell-wall-bound proteins, CWBP23 and 52. Processing of WprA exhibits a novel feature of protein export, whereby removal of the middle part of the molecule accompanies the targeting to the cell wall of its N- and C-terminal parts, which correspond to CWBP23 and 52, respectively. Sequence analyses and enzymic assays reveal that CWBP52 is a serine protease. Growth rate, cell morphology, sporulation and motility of wprA mutants apparently do not differ from those of the parent strain.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types called the prespore and the mother cell. The process is induced by nutrient deprivation and culminates with the formation of a mature spore, which is released by lysis of the mother cell. We have studied commitment to sporulation with several different assays. The results indicate that commitment occurs soon after the formation of the asymmetrically positioned division septum that separates the prespore and the mother cell. This is earlier than the previously postulated point of commitment, prespore engulfment by the mother cell. Commitment coincides approximately with activation of the early prespore- and mother-cell-specific sigma factors, σE and σF.

The expression of the Bacillus subtilis spoIVB gene, which encodes a developmental cell-cell signalling molecule, has been characterized. In some conditions, this gene can be transcribed by RNA polymerase associated with either σFor σG, in contrast to previous studies implying exclusive control by σG. However, during sporulation, only σG directs significant levels of spoIVB expression.

A proline iminopeptidase gene (pepl) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepl gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepl-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50·7, 24·5 and 33·8 kDa proteins, respectively. The ORF3-encoded Pepl protein showed 65 % identity with the Pepl proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepl gene was overexpressed in Escherichia coli. Purified Pepl hydrolysed only diand tripeptides with proline in the first position. Optimum Pepl activity was observed at pH 7·5 and 40 °C. A gel filtration analysis indicated that Pepl is a dimer of M r 53000. Pepl was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed K m and V max values of 0·8 mM and 350 mmol min−1 mg−1, respectively, and a very high turnover number of 135000 s−1.

Summary: In Lactobacillus curvatus, a phosphoenolpyruvate: mannose phosphotransferase system (mannose-PTS) has been characterized and it was shown to be involved in glucose and mannose transport, but no glucose-specific PTS activity could be detected. A 2.1 kb DNA fragment amplified by PCR from the L. curvatus genome was sequenced. Sequence analysis showed four ORFs which could encode proteins similar to PTS transporters EIIA, EIIB, EIIC and EIID of the mannose class. The expression of the manB gene (encoding EIIB) from L. curvatus in a mutant of Lactobacillus sake impaired in EIIMan activity restored this activity. Furthermore, this DNA fragment complemented the regulatory function of LevE (EIIB) in a Bacillus subtilis levE-deficient mutant, suggesting that the protein encoded by manB could also play a regulatory role in L. curvatus.