Simultaneous inactivation of the wprA and dltB genes of Bacillus subtilis reduces the yield of alpha-amylase.

AIMS: In Gram-positive bacteria, signal peptide-bearing secretory proteins are translocated through the cytoplasmic membrane and fold into their native conformation on the outside of the cell. The products of the Bacillus subtilis wprA and dltB genes separately influence post-translocational stages of the secretion process by mediating proteolytic degradation and folding of secretory proteins. Inactivation of either wprA or dltB in B. subtilis increases the yield of secretory proteins released into the culture medium in an intact and biologically active conformation. The aim of this work was to study the combined influence of these genes. METHODS AND RESULTS: A wprA/dltB double mutant was constructed, but did not have an additive effect on secretion and caused a significant reduction in the yield of alpha-amylase. CONCLUSIONS AND SIGNIFICANCE: The activities of the wprA gene and the dlt operon interact in a negative way to influence the growth cycle and protein secretion. The mechanism by which this may occur, and its potential significance for the secretion of native and non-native proteins from B. subtilis and related bacteria, is discussed.

Identification of a murine cysteinyl leukotriene receptor by expression in Xenopus laevis oocytes.

We report the identification of an EST encoding a murine cysteinyl leukotriene (mCysLT) receptor. LTD4, LTC4 and LTE4 but not LTB4 or various nucleotides activated Ca2+-evoked Cl- currents in mCysLT1 expressing Xenopus laevis oocytes. The response to LTD4 was blocked by MK-571, reduced by pretreatment with pertussis toxin (PTX), and was partly dependent on extracellular Ca2+. The identified murine CysLT1 receptor differs from the hCysLT1 receptor with regard to PTX sensitivity, receptor-mediated Ca2+ influx, and antagonist sensitivity.

The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis.

Following their secretion across the cytoplasmic membrane, processed secretory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture medium. The rate and efficiency of folding are critical in determining the yields of intact secretory proteins. The B. subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan and anionic polymers. The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage in the export process. To determine the influence of charge on late stages in the secretion of proteins from this bacterium, we have used sequence data from two related alpha-amylases, to engineer the net charge of AmyL, an alpha-amylase from Bacillus licheniformis that is normally secreted efficiently from B. subtilis. While AmyL has a pI of 7.0, chimaeric enzymes with pI values of 5.0 and 10.0 were produced and characterized. Despite the engineered changes to their physico-chemical properties, the chimaeric enzymes retained many of the enzymic characteristics of AmyL. We show that the positively charged protein interacts with the cell wall in a manner that influences its secretion.

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Lysis genes of the Bacillus subtilis defective prophage PBSX.

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are xepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show that xepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression of xhlB (encoding the putative holin) together with xlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

Interaction of the Pseudomonas cepacia DSM3959 lipase with its chaperone, LimA.

The lipA gene of Pseudomonas cepacia DSM3959 requires a downstream gene, limA, in oder to express lipase activity. The product of the lim gene, LimA, is a molecular chaperone required during the folding of lipase in oder for the lipase to adopt an active conformation. The lipase and LimA proteins have been shown to form a complex precipitable with either an anti-lipase or anti-LimA antibody. LimA has been shown to form a 1:1 complex with with prelipase and lipase isolated from "natural" P. cepacia system. The mature lipase (lacking its signal peptide) has been expressed in the presence and absence of LimA in Escherichia coli. LimA can activate mature lipase during a urea denaturation-renaturation experiment, indicating that the signal peptide is not required for the lipase to be activated by LimA. The effects of various reagents on the renaturation of lipase from 8 M urea have been examined. We propose a mechanism for the function of the LimA chaperone during the production of active extracellular lipase.

A new method for integration and stable DNA amplification in poorly transformable bacilli.

We have developed a strategy for the integration and stable amplification of DNA sequences in the chromosome of poorly transformable bacilli, which avoids the presence of a functional plasmid replication system in the integrated DNA. The parental vector for integration contains two plus origins of replication from pUB110 in the same orientation on a single plasmid. Due to the direct repeats, such plasmids produce two individual progeny vectors, one of which is dependent on the other for replication, as it lacks a functional rep gene. We have used such a progeny vector system to integrate and amplify DNA on the chromosome of Bacillus licheniformis, and show that the structure is stable in the absence of selective pressure.

Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase.

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.

A useful cloning vector for Bacillus subtilis.

We have constructed plasmid pDN1050, a new small cloning vector for Bacillus subtilis. pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.

Activation of a bacterial lipase by its chaperone.

The gene lipA of Pseudomonas cepacia DSM 3959 encodes a prelipase from which a signal peptide is cleaved during secretion, producing a mature extracellular lipase. Expression of lipase in several heterologous hosts depends on the presence of another gene, limA, in cis or in trans. Lipase protein has been overproduced in Escherichia coli in the presence and absence of the lipase modulator gene limA. Therefore, limA is not required for the transcription of lipA or for the translation of the lipA mRNA. However, no lipase activity is observed in the absence of limA. limA has been overexpressed and encodes a 33-kDa protein, Lim. If lipase protein is denatured in 8 M urea and the urea is removed by dialysis, lipase activity is quantitatively recovered provided Lim protein is present during renaturation. Lip and Lim proteins form a complex precipitable either by an anti-lipase or anti-Lim antibody. The Lim protein has therefore the properties of a chaperone.

Molecular epidemiology of antibiotic resistant Haemophilus influenzae isolated in Denmark 1981-87.

The molecular basis of antibiotic resistance was studied in 32 epidemiologically unrelated Danish clinical isolates of Haemophilus influenzae. Both non-encapsulated and capsulated type b strains were represented as well as four different biotypes. Plasmid DNA was found in 11 strains, all of which were antibiotic resistant. Antibiotic resistance was transferred to an Rd Haemophilus influenzae recipient from 5 of 6 prospective donors. Ampicillin and chloramphenicol resistance were linked markers while tetracycline resistance--when unselected--was lost in 18% of the transconjugants. Loss of Tcr was associated with loss of a plasmid DNA segment. Restriction enzyme profiles of plasmid DNA lead to the conclusion that the drug resistance plasmids are derivatives of a common, not too distant, ancestor. There is evidence of both clonal spread and horizontal transmission of related drug resistance plasmids in H. influenzae in Denmark.

Plasmids of Salmonella muenster and their relation to virulence.

The plasmid content of 104 strains of Salmonella muenster collected from bovine and human sources in Ontario from 1982 to 1984 was determined. The strains were classified into 17 different groups on the basis of the sizes of the plasmids they contained. Further division was made on the basis of the fragments obtained following digestion of the plasmids with restriction endonucleases BglI and BglII. Representative strains from each plasmid profile group were compared for resistance to serum and for virulence in mice. No differences in serum resistance or in LD50 by the oral or intraperitoneal route in mice could be associated with the presence of plasmids. Although the strains of S. muenster were recovered from clinical cases in cattle and humans, they were of low virulence for mice: the LD50 values were approximately 10,000-fold greater than that of Salmonella typhimurium.

Antibiotic resistance profiles and molecular epidemiology of Salmonella typhimurium and S. dublin, mainly from cattle.

Among 130 strains of Salmonella typhimurium and 191 strains of S. dublin, all isolated from cattle in the early 1980s, 80% and 63%, respectively, were resistant to one or more (up to three) antibiotics. Monoresistance to sulphonamides was most common. In 169 strains of the two serotypes from 1985 the antibiotic resistance load was of a similar size. The latter batch was not studied with respect to plasmids. In the strains from 1980 and 1981 the plasmid load was analysed by conjugation, transformation, extraction of plasmid DNA and subsequent electrophoresis in agarose gels. Plasmid DNA from 38 strains was further analysed by restriction with endonuclease EcoRI. On the basis of this the strains were classified into four groups: two of S. typhimurium, one of S. dublin and one group of both serotypes. This suggested that dissemination of strains and plasmids mainly occurred through clonal spread of strains.

Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon.

The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid-stabilizing activity exerted by parB. The hok gene encodes a potent cell-killing factor, and it is regulated by the sok gene product such that cells losing a parB-carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane-associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to 'stabilize' its replicon (the chromosome). The function of the relF gene is not known.

Sequence of the relB transcription unit from Escherichia coli and identification of the relB gene.

Escherichia coli relB mutants react to amino acid starvation by several abnormal responses, including accumulation of a translational inhibitor. We have isolated a relB-complementing plasmid from the Clarke and Carbon E. coli DNA library. From this plasmid we sequenced a 2140-bp segment which included the relB gene by the following two criteria: (i) it complements chromosomal relB mutations, (ii) the corresponding DNA segment cloned from chromosomal DNA of three relB mutants was defective in relB complementation. All three mutations fell within an open reading frame of 79 amino acids. A polypeptide of 9 kd compatible with this open reading frame was synthesized in maxicells and is in all probability the product of the relB gene. By nuclease S1 mapping we have determined the transcription start and stop of an 870 base transcript of the relB gene.

Prevalence and molecular epidemiology of antibiotic-resistant Salmonella typhimurium and Salmonella dublin in Danish cattle.

Among 130 strains of S. typhimurium and 191 strains of S. dublin, all from cattle, 80% and 63%, respectively, were resistant to one, two or three antibiotics. Mono-resistance to sulphonamides was most common. Plasmid load was analysed by conjugation, transformation, extraction of plasmid DNA and subsequent electrophoresis in agarose gels. Plasmid DNA from 38 strains was further analysed by restriction with endonuclease EcoR1. On the basis of this, the strains were classified into four groups. Two groups held S. typhimurium, one held S. dublin and one group held both serotypes. This suggests that dissemination of strains and plasmids mainly occurs through clonal spread of strains. However, plasmid transfer per se also takes place, as exemplified by the fact that indistinguishable plasmids were found in two different serotypes. Strains of one group had contaminated a water-course. Strains of this group were furthermore isolated from humans in the same area as the infected cattle. Strains of this group had an R pattern and a phage-type similar to the R pattern and phage-type of the early isolates of a strain that became epidemic in British cattle. It is discussed whether Denmark, which was previously almost free of cattle salmonellosis, is experiencing the first warnings of an epidemic similar to the one in the UK.

Relatedness of chloramphenicol resistance plasmids in epidemiologically unrelated strains of pathogenic Escherichia coli from man and animals.

I have examined 20 plasmids conferring chloramphenicol resistance (Cm) in multiresistant strains of Escherichia coli pathogenic for man and piglets. In Denmark, one plasmid family, exemplar pHG33, is responsible for all chloramphenicol resistance in serotypes of E. coli found in diseased piglets. A closely related plasmid, pHG50, was identified in an enteropathogenic E. coli (EPEC) strain from an infant. The isolate was epidemiologically unrelated to the piglet isolates. The molecular relatedness of the plasmids was established by restriction enzyme analyses and Southern blots. Chloramphenicol resistance plasmids in E. coli from urinary tract infections, or in English EPEC strains, did not show the same close relatedness with the piglet plasmid pHG33. However, many were of the same incompatibility group and their restriction profiles displayed a number of common bands. The close relatedness of pHG50 and pHG33 suggests exchange of plasmids between pathogenic serotypes of E. coli from man and animals. The infant from whom the EPEC strain carrying plasmid pHG50 was isolated might have acquired it from piglets. Disease in human babies caused by EPEC strains is now rare in Denmark and no Cm-resistant strains are found. Possible reasons for the loss of Cm-resistance plasmids from human strains and their retention in piglet strains are discussed.

Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets.

All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers. Two strains from 1978 carried plasmids with similar characters. Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups. These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types. It is concluded that these plasmids have evolved from a single plasmid.

New translocation sequence mediating tetracycline resistance found in Escherichia coli pathogenic for piglets.

A discrete piece of deoxyribonucleic acid coding for tetracycline (Tc) resistance was found to move from one R plasmid to another in an Escherichia coli strain which is pathogenic for piglets. Since this phenomenon took place also in rec strains, the Tc segment was classified as a transposon and called Tn804. Restriction enzyme analysis with EcoRI, BglII, and HindIII indicated that Tn804 is related to Tn10, a well-known transposon that codes for resistance to tetracycline. Hybridization between plasmids carrying the two transposons provided proof of homology between Tn10 and prt of Tn804. Electron microscopic studies showed a transposon-like structure composed of one loop-stem structure with inverted repetitions of approximately 0.9 megadaltons inserted into the loop of a second loop-stem structure. It is suggested that Tn804 is composed of Tn10 plus another transposable sequence.