摘要:

SummaryTheChlamydia trachomatismajor outer membrane protein (MOMP) is the quantitatively predominant surface protein which has important functional, structural and antigenic properties. We have cloned and overexpressed the MOMP inEscherichia coliThe MOMP is surface exposed inC. trachomatisand capable of eliciting protective antibodies in infected hosts, and therefore has potential as a candidate vaccine to prevent infection with this significant human pathogen. The recombinant MOMP clone, L2rMOMP, contained the entire MOMP gene including the encoded leader sequence. Large quantities of chlamydial MOMP were expressed, some of which was processed and translocated to theE. colisurface. Surface localization of the MOMP was demonstrated by the binding of anti‐MOMP monoclonal antibodies to the surface of the induced clone, and was visualized by fluorescence and electron microscopy. The induction of MOMP expression had a rapidlylethaleffect on the L2rM0MPE. coliclone. Although no genetic system exists forChlamydia, development of a stable, inducibleE. colidone which overexpresses the chlamydial MOMP permits a study of the biological properties of the MOMP, including the contribution of the MOMP variable segments to the topographical interactions which determine the antigenic structure responsible for human immune respons

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01545.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryMany natural DNA sequences are restricted inEscherichia coliK‐12, not only by the classic Type I restriction systemEcoK, but also by one of three modification‐specific restriction systems found in K‐12. The McrBC system is the best studied of these. We infer from the base composition of themcrBCgenes that they were imported from an evolutionarily distant source. The genes are located in a hypervariable cluster of restriction genes that may play a significant role in generation of species identity in enteric bacteria. Restriction activity requires the products of two genes for activity bothin vivoandin vitro.ThemcrBgene elaborates two protein products, only one of which is required for activityin vitro, but both of which contain a conserved amino acid sequence motif identified as a possible GTP‐binding site. ThemcrCgene product contains a leucine heptad repeat that could play a role in protein‐protein interactions. McrBC activityin vivoandin vitrodepends on the presence of modified cytosine in a specific sequence context; three different modifications are recognized. Thein vitroactivity of this novel multi‐subunit restriction enzyme displays an absolute requirement for GTP as

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01546.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryTurnover of the chloramphenicol acetyltransferase (cat) messenger RNA inEscherichia coliwas investigated by analysis of cellular mRNA decay intermediates and the transcript sequence. Analysis of the sequence has revealed the presence of a repetitive extragenic palindromic (REP) element at the 3′ end of the transcript as well as several 5′‐UUAU‐3′ sequences, two elements which have roles in modulating turnover of otherE. colimRNAs. ForcatmRNA, however, removal of the REP sequence has no effect on the half‐life of the transcript, indicating that the REP sequence does not stabilize the upstream region of this message. Results from mapping of the mRNA decay products by several techniques suggest that the message is instead subject to endonucleolytic attack at five sites 5′ of the REP element. The sequence UUAU is present at three of these five sites. It also appears that thecatmRNA is sequentially cleaved in a 3′ to 5′ direction during turnover of this mRNAin vivo.A model forcatmRNA turn

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01547.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryA method is described to determine simultaneously the effect of any changes in the ribosome‐binding site (RBS) of mRNA on translational efficiency inBacillus subtilisandEscherichia coli in vivo.The approach was used to analyse systematically the influence of spacing between the Shine‐Dalgarno sequence and the initiation codon, the three different initiation codons, and RBS secondary structure on translational yields in the two organisms. BothB. subtilisandE. coliexhibited similar spacing optima of 7–9 nucleotides. However,B. subtilistranslated messages with spacings shorter than optimal much less efficiently thanE. coli.In both organisms, AUG was the preferred initiation codon by two‐ to threefold. InE coliGUG was slightly better than (JUG while inB. subtilisUUG was better than GUG. The degree of emphasis placed on initiation codon type, as measured by translational yield, was dependent on the strength of the Shine‐Dalgarno interaction in both organisms.B. subtilisv/as also much less able to tolerate secondary structure in the RBS thanE. coli.While significant differences were found between the two organisms in the effect of specific RBS elements on translation, other mRNA components in addition to those elements tested appear to be responsible, in part, for translational species specificity. The approach described provides a rapid and systematic means of elucidating such additional det

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01548.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummarySubtilisins are extracellular sery l‐proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N‐terminal extensions (pro‐regions) which have also been identified in several other proteases (Silenet al., 1988; Vasanthaet al., 1984; Polhneret al., 1987; Hendersonet al., 1987; Yanagidaet al., 1986; Takagiet al., 1985). The pro‐region holds the pro‐protease associated with the membrane and release of the protease takes place as a result of pro‐region removal by autocatalytic processing (Egnell and Flock, 1991).In this report we describe the construction of four deletion‐mutations in the gene encoding subtilisin Carlsberg at the junction between the pro‐region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro‐regionin cis, whereas cleavage by exogenous subtilisin could still occurin trans.Point mutations were also introduced in positions ‐5 to +4 around the pro‐region and native subtilisin cleavage site. Processing of pro‐subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro‐region is independent of the amino acid sequence

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01549.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThexcpgenes are required for the secretion of most extracellular proteins byPseudomonas aeruginosa.The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periptasm via a signal sequence‐dependent pathway. To date, analysis of threexcpgenes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, thexcpAgene was shown to be identical topilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven otherxcpgenes, designatedxcpRto‐X, are presented. TheNtermini of four of the encoded Xcp proteins display similarity to theN‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstratedin vivo.Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide‐binding site, ATP hydrolysis may provide energy for bot

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01550.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe promoter region of the agarase gene (dagA) ofStreptomyces coelicolorA3(2) is complex; it consists of four distinct promoters with different ‐10 and ‐35 regions. We report the isolation of a form of RNA polymerase that mediates transcriptionin vitrofrom thedagAp4 promoter. The core components of this RNA polymerase are associated with a polypeptide ofc.66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from thedagAp4 andBacillus subtilis vegpromotersin vitro.Alignment of the DNA sequences of these two promoters shows that they have bases in common in the ‐10 and ‐35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria.N‐terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of thehrdBgene. Previous experiments showed that the predicted amino acid sequence of thehrdBgene product is very similar to the major sigma factors of other bacteria and suggested that disruption of thehrdBgene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase ofStreptomyces coelicolorA3(2).We have developed an improved protocol for the renaturation of sigma factors that have been Isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01551.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryThe unit‐copy P1 plasmid depends for stability on a plasmld‐encoded partition region calledpar, consisting of theparAandparBgenes and theparSsite. ParA is absolutely required for partition, but its partition‐critical role is not known. Purified ParA protein is shown to possess an ATPase activityin vitrowhich is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression ofparAandparB, and purified ParA has an ATP‐dependent, site‐specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The rote of the ATP‐dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, i

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01552.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryMycoplasma pulmonisis a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high‐frequency phenotypic switching have been reported forM. pulmonis, the molecular basis of which is unknown. We report here that during growth theM. pulmonischromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred inM. pulmonisindicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infection

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01553.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SummaryFull elastolytic activity inPseudomonas aeruginosais a result of the combined activities of elastase, alkaline proteinase, and thelasAgene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA‐mediated enhancement of elastotysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave β‐casein. Preliminary analysis of β‐casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine p

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1992.tb01554.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY