Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of alpha-amylase in Bacillus subtilis.

Regulated expression of AmyQ alpha-amylase of Bacillus amyloliquefaciens was used to examine the capacity of the protein secretion apparatus of B. subtilis. One B. subtilis cell was found to secrete maximally 10 fg of AmyQ per h. The signal peptidase SipT limits the rate of processing of the signal peptide. Another limit is set by PrsA lipoprotein. The wild-type level of PrsA was found to be 2 x 10(4) molecules per cell. Decreasing the cellular level of PrsA did not decrease the capacity of the protein translocation or signal peptide processing steps but dramatically affected secretion in a posttranslocational step. There was a linear correlation between the number of cellular PrsA molecules and the number of secreted AmyQ molecules over a wide range of prsA and amyQ expression levels. Significantly, even when amyQ was expressed at low levels, overproduction of PrsA enhanced its secretion. The finding is consistent with a reversible interaction between PrsA and AmyQ. The high cellular level of PrsA suggests a chaperone-like function. PrsA was also found to be essential for the viability of B. subtilis. Drastic depletion of PrsA resulted in altered cellular morphology and ultimately in cell death.

Picornavirus proteins share antigenic determinants with heat shock proteins 60/65.

Immunological cross-reactions between enteroviruses and islet cell autoantigens have been suggested to play a role in the etiopathogenesis of insulin dependent diabetes mellitus (IDDM). In the nonobese diabetic mouse, an autoimmune model of IDDM, one of the reactive beta cell autoantigens is the heat shock protein 60 (HSP60). These studies were prompted by sequence homology discovered between the immunogenic region in HSP60 and two regions in enterovirus capsid proteins, one in the VP1 protein and the other in the VP0, the precursor of VP2 and VP4 proteins. Possible immunological cross-reactions between enterovirus proteins and heat shock proteins were studied by EIA and immunoblotting by using purified virus preparations, viral expression proteins VP1 and VP0, and recombinant HSP60/65 proteins, and corresponding polyclonal antisera. The HSP60/65 family of proteins is highly conserved and there is a striking degree of homology between bacterial and human heat shock proteins. Rabbit antibodies to HSP65 of Mycobacterium bovis that reacted with human HSP60 were also found to recognise capsid protein VP1 of coxsackievirus A9, VP1, and/or VP2 of coxsackievirus B4. Both viruses were also recognised by antisera raised against HSP60 of Chlamydia pneumoniae. In addition to the capsid proteins derived from native virions, antisera to both bacterial HSP proteins recognised expression protein VP1 of coxsackievirus A9. The cross-reactivity was also demonstrated the other way around; antisera to purified virus particles reacted with the HSP 60/65 proteins to some extent. These results suggest that apart from the well-documented sequence homology between the 2C protein of coxsackieviruses and the beta-cell autoantigen glutamic acid decarboxylase, there are other motifs in picornavirus proteins homologous to islet cell autoantigens, which might induce cross-reacting immune responses during picornavirus infections.

Exopolysaccharide-producing bacteria from sugar beets.

Six hundred microorganisms were isolated from sugar beets collected from different parts of Finland to study their slime production. A total of 170 of them produced exopolysaccharides, of which 35% were heteropolysaccharides. The yield of heteropolysaccharides from sucrose was lower than that of dextrans. Five isolates, which were chosen for closer study, were identified as Leuconostoc mesenteroides (two species), Rahnella aquatilis (two species), and Enterobacter amnigenus.

Antibodies to meningococcal class 1 outer-membrane protein and its variable regions in patients with systemic meningococcal disease.

Antibodies to the meningococcal serosubtype-specific P1.7,16 protein and its variable regions (VR) were analysed in 28 convalescent sera drawn 8-36 months after systemic meningococcal disease by immunoblotting and enzyme immunoassay (EIA) methods. EIA antigens were the meningococcal P1.7,16 protein, produced in Bacillus subtilis, and peptides covering its VR1 (P1.7 region) and VR2 (P1.16 region) inserted into a bacterial penicillinase protein. In the immunoblotting method, three meningococcal reference strains were used; they expressed either the P1.7,16 protein, or only its VR1 or VR2 epitopes in their class 1 proteins. Both methods showed a strong IgG response in four sera to P1.7,16 and VR2, but not to VR1; 18 sera had no or weak anti-class 1 protein activity. The six remaining sera were positive only on blots. The VR2-specific sera had 30-fold higher bactericidal activity than those with negligible P1.7,16 responses. Previous vaccination of the patients with a B:15:P1.7,16 meningococcal vaccine was associated with a strong anti-P1.7,16 and anti-VR2 booster response that declined with time. The subtype-specific antibody activity in some sera indicated colonisation after disease by meningococci with class 1 proteins different from the strain that had caused disease.

Molecular cloning and analysis of the yeast flocculation gene FLO1.

The DNA sequence of the flocculation gene FLO1 of Saccharomyces cerevisiae, which is located on chromosome I (Watari et al., 1989) was determined. The sequence contains a large open reading frame (ORF) of 2586 bp and codes for a protein of 862 amino acids. However, further study (genomic Southern and polymerase chain reaction analyses) indicated that the gene we cloned was not the intact FLO1 gene but a form with an approximately 2 kb deletion in the ORF region. The intact FLO1 gene was then cloned and its nucleotide sequence determined. The sequence revealed that the ORF of the intact gene is composed of 4611 bp which code for a protein of 1537 amino acids. A remarkable feature of the putative Flo1 protein is that it contains four families of repeated sequences composed of 18, 2, 3 and 3 repeats and that it has a large number of serines and threonines. In the deleted FLO1 form, a large part of these repeated sequences was missing. The N- and C-terminal regions are hydrophobic and both contain a potential membrane-spanning region, suggesting that the Flo1 protein is an integral membrane protein and a cell wall component.

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

Xylitol production by recombinant Saccharomyces cerevisiae.

We obtained efficient conversion of xylose to xylitol by transforming Saccharomyces cerevisiae with the gene encoding the xylose reductase (XR) of Pichia stipitis CBS 6054. Comparison of the chromosomal and cDNA copies of the XYL1 gene showed that the genomic XYL1 contains no introns, and an XR monomer of 318 amino acids (35,985 D) is encoded by an open reading frame of 954 bp. The amino acid sequence of the P. stipitis XR is similar to several aldose reductases, suggesting that P. stipitis XR is part of the aldoketo reductase superfamily. S. cerevisiae transformed with the XYL1 gene gave over 95% conversion of xylose into xylitol, a yield not obtainable with natural xylose utilizing yeasts.

Immunogenicity and protective efficacy of pertussis toxin subunit S1 produced by Bacillus subtilis.

Pertussis toxin (PT) subunit S1 was produced in Bacillus subtilis as a secretory protein designated BacS1. BacS1 was partially purified and used to immunize mice. The sera were tested for PT-neutralizing antibodies and for protective capacity in a mouse model. Unlike previous findings with recombinant S1 from Escherichia coli, the recombinant BacS1 protein induced antibodies that were both neutralizing and protective. An adjuvant was necessary for efficient immunization with BacS1 but not with PT. Of the four adjuvants tested, aluminium phosphate gel was insufficient whereas Freund's incomplete adjuvant, Klebsiella lipopolysaccharide and Ribi's monophosphoryl lipid A-trehalose dimycolate emulsion all resulted in protective antibody production in NIH mice.

Production and secretion of pertussis toxin subunits in Bacillus subtilis.

Pertussis toxin (PT) is a major component of today's acellular whooping cough vaccines. The use of acellular vaccines is predicted to increase sharply in the near future. There is therefore a need to produce PT in a way that makes its purification as easy as possible. Our approach was to express all five PT subunits individually in Bacillus subtilis. We have used vectors containing the promoter and signal sequences of the alpha-amylase gene of Bacillus amyloliquefaciens followed by an insert encoding the appropriate PT-subunit. All PT-subunits were secreted and found in the culture supernatant. The level of expression varied considerably: S1 and S5 were produced in large quantities whereas much smaller amounts of S2, S3 and S4 were found. The subunits were also present in the membrane fraction of the respective strains.