摘要:

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed‐function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface‐exposed receptors dedicated to binding haem and haem‐proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB‐dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is pr

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030383.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The genekdtAofChlamydia pneumoniaestrain TW‐183, encoding the enzyme 3‐deoxy‐α‐d‐manno‐octulosonic acid (Kdo)transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA ofChlamydia trachomatisandChlamydia psittaci. The gene was expressed in the Gram‐positive hostCorynebacterium glutamicumand the primary gene product was characterized as a multi‐functional glycosyltransferase. Cell‐free extracts generatedin vitrothe genus‐specific epitope ofChlamydiacomposed of the trisaccharide (αKdo(2–8)αKdo(2–4)αKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: ‘one

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030391.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Lysophosphatidic acid (LPA) acyltransferases ofNeisseria meningitidisandNeisseria gonorrhoeaewere identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. InEscherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916‐generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916insertion in the meningococcal mutant was localized within a 771 bp open reading frame. The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. InN. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designatednIaA(neisserialLPA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity,>40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcalnIaAgene complemented intransa temperature‐sensitiveE. coliPlsCts−mutant. Tn916and Ω‐cassette insertional inactivations of the neisserialnIaAgenes altered the membrane glycerophospholipid compositions of bothN. meningitidisandN. gonorrhoeaebut were not lethal. Therefore, the pathogenicNeisseriaspp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcalnIaAmutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcalnIaAmutant, was increased in the meningococcalnIaAmutant. We postulate that the pathogenicNeisseriaspp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence ofnIaAenzymatic activity.Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on cap

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030401.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Listeria monocytogenesandShigella flexneriare two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein inL. monocytogenesand the lcsA (VirG) surface protein inS. flexneri. ActA and lcsA share no sequence homology. To assess the role of the two proteins in the generation of actin‐based movement, we expressed them in the genetic context of two non‐actin polymerizing, non‐pathogenic bacterial species,Listeria innocuaandEscherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts fromXenopuseggs, as visualized by the formation of characteristic actin comet tails.E. coliexpressing lcsA moved about two times faster thanListeriaand displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half‐lives) were similar in lcsA‐ and ActA‐induced actin tails suggesting that by using unrelated surface molecules,L. monocytogenesandS. flexnerimove intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduct

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030413.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The intracellular bacterial pathogenListeria monocytogenesmoves inside the host‐cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin‐driven bacterial propulsion, we have now generated severalL. monocytogenesstrains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino‐terminal part is critical for F‐actin assembly and movement. The internal proline‐rich repeats and the carboxy‐terminal domains are not essential. However,in vitromotility assays have demonstrated that mutants lacking the proline‐rich repeats domain of ActA moved two times slower (6±2 µm min−1) than the wild type (13±3µm min−1}). In addition, phosphatase treatment of protein extracts of cells infected with theL. monocytogenesstrains expressing the ActA variants suggested that phosphorylation may not be essent

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030425.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The gene for the Tn10Tet repressor (TetR) was subjected to deletion mutagenesis. Screening for a transdominant operator‐binding negative phenotype yielded 10 mutants with internal deletions. Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the α‐helix‐turn‐α‐helix DNA‐binding motif. Five deletions range from residue K84 to residues between R87 and K98. Since residues from the N‐terminus up to position 98 are not necessary for dimerization, this must take place in the C‐terminal half of the protein. Ability to dimerize was probed by introducing ochre non‐sense codons (oc) at residues G138, H151, E159, l174, or K202. Koc202 shows wild‐typein vivooperator‐binding and inducibility by tetracycline indicating that the six C‐terminal residues of TetR are not important for activity. Mutants with longer C‐terminal truncations are inactive and not transdominant. They show reduced steady‐state protein levels and are probably impaired in folding and degradedin vivo. Two mutants (Δ151–166, Δ164–166) with deletions in a region variable in primary structure and length among Tet repressers from different resistance determinants bindtetoperator efficiently, but are not inducibie by tetracycline. This result indicates that these residues are not important for dimer for

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030437.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The Flp site‐specific recombinase is encoded by the 2 µm plasmid ofSaccharomyces cerevisiaeand is a member of the integrase family of recombinases. Like all members of the integrase family studied, Flp mediates recombination in two steps. First, a pair of strand exchanges creates a Holliday‐like intermediate; second, this intermediate is resolved to recombinant products by a second pair of strand exchanges.Evidence derived from experiments using linear substrates indicates that Flp's active site is composed of two Flp protomers. One binds to the Flp recognition target site (FRT site) and activates the scissile phosphodiester bond for cleavage. Another molecule of Flp bound elsewhere in the synaptic complex (in trans) donates the nucleophilic tyrosine that executes cleavage and thereby becomes covalently attached to the 3′ phosphoryl group at the cleavage site.It has previously been shown that Flp efficiently resolves synthetic, Holliday‐like (χ) structures to linear products. In this paper, we examined whether resolution of χ structures by Flp also occurs via thetranscleavage mechanism. We usedin vitrocomplementation studies of mutant Flp proteins as well as nicked χ structures to show that Flp resolves χ structures bytranscleavage. We propose a model for Flp‐mediated recombination that incorporatestranscleavage at both the initial and resolution steps of s

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030449.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation inBacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic‐like process. Surprisingly, the chromosome of the developing spore and the small, acid‐soluble proteins, known as α/β‐type SASPs, that are known to coat the spore chromosome, were found to co‐localize to a doughnut‐like ring of approximately 1 µm in diameter. The use of a double mutant lacking the α/β‐type SASP demonstrated that these high abundance, DNA‐binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation inB. subtilisis a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030459.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

The β recombinase is unable to mediatein vitroDNA recombination between two directly oriented recombination sites unless a bacterial chromatin‐associated protein (Bacillus subtilisHbsu orEschrichia coliHU) is provided. By electron microscopy, we show that the role of Hbsu is to help in joining the recombination sites to form a stable synaptic complex. Some evidence supports the fact that Hbsu works by recognizing and stabilizing a DNA structure at the recombination site, rather than by serving as a bridge between β recombinase dimers through a protein‐protein interaction. We show that the mammalian HMG1 protein, which shares neither sequence nor structural homology with Hbsu, can also stimulate β‐mediated recombination. These chromatin‐associated proteins share the property of binding to DNA in a relatively non‐specific fashion, bending it, and having a marked preference for altered DNA structures. Hbsu, HU or HMG1 proteins probably bind specifically at the crossing‐over region, since at limiting protein‐DNA molar ratios they could not be outcompeted by an excess of a DNA lacking the crossing over site. Distamycin, a minor groove binder that induces local distortions in DNA, did not affect the binding of β protein to DNA, but inhibited the formation of the

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030471.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Salmonella typhimuriumsecreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes (sspB,sspC,sspD, andsspA), located betweenspaTandprgH, which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous toShigella flexnerisecreted proteins lpaB, lpaC, lpaD and lpaA. A non‐invasive mutant with a transposon insertion insspClacks Ssp of 87,42 and 36 kDa. Complementation analyses show thatsspCandsspDencode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded bysspA.sspCandsspD, but notsspAare required for invasion. Amino‐terminal sequencing shows that SspC and SspA are secreted without amino‐terminal processing. We further demonstrate that Ssp secretion requires proteins encoded byprgHIJK, homologous to theShigellalpa secretion system, since SspA is abundantly secreted by wild‐type bacteria but is completely retained within the cellular fraction of aprgHIJKmutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild‐type bacteria. These data indicate that major secreted invasion determinants ofS. typhimuriumare structurally and functionally homolgous toS. flexnerilpa

ISSN:0950-382X    

DOI:10.1111/j.1365-2958.1995.mmi_18030479.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY