bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene
Authors: Chen Yi‐Chi , Shipley Gregory L. , Ball Timothy K. , Benedik Michael J.
Published: October 2006

The extracellular nuclease of Serratia marcescens is regulated in a complex fashion. Unlike most catabolic enzymes, it appears not to be substrate regulated. However we have shown it to be regulated by an SOS‐like system in S. marcescens. Additionally nuclease expression is regulated In a growth‐phase‐dependent manner. In this work we demonstrate that growth‐phase‐dependent regulation is at the transcriptional level. The putative LexA‐binding site which mediates SOS regulation is shown to act as an operator site in viva. The boundaries of a minimal promoter, stilt regulated by growth phase and SOS regulation, are defined along with the transcriptional start site. However, a region upstream of the nuclease promoter is shown to enhance significantly the expression of nuclease.
Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities
Authors: Srikumar R. , Chin A. C. , Vachon V. , Richardson C. D. , Ratcliffe M. J. H. , Saarinen L. , Käyhty H. , Mäkelä P. H. , Coulton J. W.
Published: October 2006

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial ceil surface, seven mouse monoclonal anti‐Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104–139; domain (ii) amino acids 162–174; and domain (iii), amino acids 267–341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement‐mediated tysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface‐binding, they were neither bactericidal nor protective.
Microtubule assembly and phage morphogenesis: new results and classical paradigms
Authors: Weinstein Brant , Solomon Frank
Published: October 2006

The classical analyses of phage morphogenesis provide experimental paradigms for dissecting other assembly pathways. A new set of results, derived from examination of the quantitative controls of tubulin levels and microtubule assembly in Saccharomyces cerevisiae, evokes the balance of components’ hypothesis. These results show that balanced levels of the tubulin proteins are crucial for microtubule assembly. Imbalances leading to excess beta tubulin have far more deleterious consequences than those leading to excess alpha tubulin, including dramatic cellular toxicity, quantitative depolymerization of cellular microtubules, and self‐aggregation of the excess beta tubulin. These and other results suggest that beta tubulin may possess a unique ability to interact with a component of microtubule nucleating sites, and provide a rationale for the universal polarity of nucleated microtubules.
Elongation factor Tu: a molecular switch in protein biosynthesis
Authors: Weijland Albert , Harmark Kim , Cool Robbert H. , Anborgh Pieter H. , Parmeggiani Andrea
Published: October 2006

Elongation factor Tu (EF‐Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide‐binding protein that in the ‘on’ state acts as a carrier of amino acyl‐tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co‐ordinated application of biochemical, physico‐chemical and genetic methods. Crystallographic analysis at 2.6 Å resolution and site‐directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide‐binding domains of EF‐Tu and human H‐ras p21 protein. The regulation of the expression of the two EF‐Tu‐encoding genes in E. coli, particularly that of tufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF‐Tu in the elongation cycle have been reinvestigated, leading to new insights. These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF‐Tu in the bacterial cell.
Establishment of cell‐specific transcription during sporulation in Bacillus subtilis
Authors: Errington J. , Illing N.
Published: October 2006

One of the most intriguing questions posed by bacterial spore formation concerns the establishment of cell‐specific gene expression in the prespore and mother cell. Recent results now suggest that sigma factors, in addition to their temporal roles in the control of gene expression, may also be the key determinants of differential gene expression during sporulation in Bacillus subtilis. The genes encoding two sporulation‐specific sigma factors, σE and σF, are expressed soon after the initiation of sporulation, before the formation of the spore septum that separates the prespore and mother cell compartments, it now appears that σE and σF direct transcription only after septation and then In a specific cell type, suggesting that the segregation of the sigma activities after septation is a key event in the establishment of differential gene expression. The mechanism responsible for this segregation is complex, involving at least seven other gene products. We discuss possible models for the interactions between the sigma factors and the establishment of cell‐specific transcription.
A key role for type 1 pili in enterobacterial communicability
Authors: Bloch Craig A. , Stocker Bruce A. D. , Orndorff Paul E.
Published: October 2006

Up to 80% of faecal Escherichia coil strains are able to produce type 1 pili. These filamentous bacterial surface organelles, which mediate mannose‐sensitive attachment to mammalian epithelial cells, are also conserved throughout the Enterobacteriaceae. As a potential explanation for their prevalence among intestinal isolates of enteric bacteria, it has been widely speculated that type 1 pili are important for adherence to the host's intestinal mucosa. However, conclusive evidence for this idea is lacking, and there are reasonable grounds for doubting such an effect. Permanent interruption of type 1 pillation in previously pil+E. coli (by directed mutagenesis of pilA, the gene coding for the major structural subunit of type 1 pili) does not diminish the density of intestinal colonization in individual animals. Rather, as we demonstrate here, this lesion results in a dramatic decrease in transmission of E. coli K1 from experimentally colonized neonatal rats to their littermates. The enhanced communicability associated with type 1 pillation suggests a heretofore unrecognized explanation for the prevalence of type 1 pili among intestinal E. coli; one that does not necessarily require the direct action of these organelles at the intestinal mucosa.
An amino‐proximal domain required for the localization of FtsQ in the cytoplasmic membrane, and for its biological function in Escherichia coli
Authors: Dopazo Ana , Palaclos Pilar , Sánchez Manuel , Pla Jesús , Vicente Miguel
Published: October 2006

The location of FtsQ, an Escherichia coli protein essential for cell division, is, under physiological conditions, in the cytoplasmic membrane facing towards the periplasmic space. An amino‐proximal hydrophobic domain is required for FtsQ to reach its location and for its activity in the cell. Overexpression of modified forms of FtsQ is deleterious for the cell.
The interaction of naturally elaborated blebs from serum‐susceptible and serum‐resistant strains of Neisseria gonorrhoeae with normal human serum
Authors: Pettit Robin K. , Judd Ralph C.
Published: October 2006

We studied the interaction of normal human serum immunoglobulins with outer‐membrane bleb antigens of Neisseria gonorrhoeae. Gonococcal 68000 Dalton and Lip (H.8 antigen) outer‐membrane proteins were recognized by normal human serum immunoglobulins in blebs from serum‐resistant strains, but not in blebs from serum‐susceptible strains. The addition of blebs from a serum‐resistant strain to bactericidal assays resulted in significantly greater inhibition of serum killing than the addition of blebs from a serum‐susceptible strain. Our results indicate that blebs from two serum‐resistant gonococcal strains have an enhanced ability to bind and remove cell‐targeted bactericidal factors, and that outer‐membrane bleb‐bing may contribute to serum resistance.
The excC gene of Escherichia coli K‐12 required for cell envelope integrity encodes the peptidoglycan‐associated lipoprotein (PAL)
Authors: Lazzaroni Jean‐Claude , Portalier Raymond
Published: October 2006

The excC mutants of Escherichia coli are hypersensitive to drugs such as cholic acid and release periplasmic proteins Into the extracellular medium. A 1884 bp fragment carrying the excC gene was isolated and sequenced. It contains the 3′ end of the tolB gene which maps at min 17 on the E. coll map and an open reading frame which encodes the 18748 Da ExcC protein. The protein is composed of a hydrophobic region of 22 residues and displayed an overall hydrophilic configuration. It was shown that the ExcC protein is indeed the PAL (peptidoglycan‐associated lipoprotein) described by Mizuno (1979). The pal gene had not yet been characterized on the E. coli linkage map since no obvious phenotype could be identified for mutations in this gene. A topologic analysis of the PAL protein using PAL–PhoA translational fusions showed that PAL is associated with the outer membrane only by its N‐terminal moiety. The carboxy‐terminal part of the protein is necessary for correct interaction of PAL with the peptidoglycan layer.
Cobalamin (vitamin B12) repression of the Cob operon in Salmonella typhimurium requires sequences within the leader and the first translated open reading frame
Authors: Richter‐Dahlfors Agneta A. , Andersson Dan I.
Published: October 2006

Expression of the Cob operon in Salmonella typhimurium is repressed by cobalamin (Cbl). Here it is shown that Cbl repression is mediated by a post‐transcriptional regulatory mechanism that requires sequences within the leader and the first translated open reading frame, the cbiA gene. Transcriptional and translational Cob::lacZ fusions containing various lengths of Cob DNA were analysed. In a translational Cob::lacZ fusion 407 bp of leader sequence (+69 to +476) was sufficient to confer normal repression. However in a transcriptional Cob::lacZ fusion a 618 bp region (+69 to +687) was required for normal repression. This 618 bp region included sequences in the leader as well as sequences within the cbiA gene. Point mutations which resulted in loss of repression control were isolated and shown to be clustered in the leader sequence (+257 to +380). This region contains a putative hairpin‐loop structure which we propose functions as an RNA operator site for a vitamin B12‐responsive repressor protein.
Differential levels of fertility inhibition among F‐like plasmids are related to the cellular concentration of finO mRNA
Authors: van Biesen T. , Frost L. S.
Published: October 2006

The FinOP system of F‐like plasmids consists of an antisense RNA (FinP) and a 22 kDa protein (FinO) which act in concert to prevent the translation of TraJ, the positive regulator of the transfer operon. Earlier studies suggested that two different variants of finO were responsible for differential levels of fertility inhibition among F‐like plasmids. We have shown that these variations are due to the presence of an additional open reading frame (orf286) upstream of the finO gene of conjugative plasmids that are highly repressed for transfer. When orf286 and finO are linked in cis, the level of FinO expression is increased because of a rise in the cellular concentration of finO mRNA. orf286 frameshift and deletion mutants also gave the same concentration of finO transcript, suggesting that the effect is due to mRNA stabilization. We suggest that the levels of fertility inhibition exhibited by F‐like plasmids are a function of their cellular FinO concentration.
Processing of a polycistronic mRNA requires a 5′cis element and active translation
Authors: Alifano P. , Piscitelli C. , Blasi V. , Rivellini F. , Nappo A. G. , Bruni C. B. , Carlomagno M. S.
Published: October 2006

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild‐type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis‐acting element and translation of the cistron immediately downstream of the 5′ end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.
What is known about the structure and function of the Escherichia coli protein FirA?
Authors: Dicker Ira B. , Seetharam Shobha
Published: October 2006

The firA gene is essential for growth of Escherichia coli growth and lies in the 4‐minute region of the genome. firA encodes the FirA protein which contains 341 amino acids and has an apparent molecular mass of 36 kDa. Genetic evidence suggests that FirA plays a role in transcription since certain firA alleles confer temperature sensitivity for growth and RNA synthesis as well as reversing the rifampin resistance of rifampin‐resistant rpoB mutants (‘fir’ effect). FirA co‐immunoprecipitates with RNA polymerase holoenzyme, implying a physical association with the transcriptional machinery, possibly with the beta subunit of RNA polymerase. FirA contains a previously undescribed isoleucine/valine‐rich six‐amino‐acid repeat occurring 14 times within the N‐terminal and 12 more times within the C‐terminal half of the protein. This repeat can be formulated as [HXXXhZ]n with ‘H’ representing a large non‐polar residue (usually isoleucine),‘h’ representing a smaller non‐polar residue, Z’ representing either charged/polar or aromatic residues, XXX representing residues typical of beta turns, and ‘n’ being equal to the repeat number. We refer to this repeat as an isoleucine patch. Proteins encoded by three E. coli acyltransferases also contain this motif which is roughly positioned in each case, within the amino‐ and carboxyl termini, as in FirA. When the sequences of these proteins are aligned, a region of poor similarity separates the isoleucine patches. The significance of these repeats remains unknown although we speculate that they play an important structural role in the organization and function of FirA (and other proteins containing isoleucine patches), possibly by acting as homo‐ or hetero‐dimerization interfaces. This is supported by the finding that mutations conferring temperature sensitivity for growth or both temperature sensitivity for growth as well as the ‘fir’ effect, map to hypothetical turn residues within the repeats of FirA.
The suicidal DNA repalr methyltransferases of microbes
Authors: Samson Leona
Published: October 2006

Virtually every organism so far tested has been found to possess an extremely efficient DNA repalr mechanism to ensure that certaln alkylated oxygens do not accumulate in the genome. The repalr is executed by DNA methyltransferases (MTases) which repalr DNA O6‐methylguanine (O6MeG), O4‐methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repalr of just one, or sometimes two, O‐alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection agalnst death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well‐characterized microbial DNA repalr MTases.
Phosphorylation of IcsA by cAMP‐dependent protein kinase and its effect on intercellular spread of Shigella flexneri
Authors: D'Hauteville Hélène , Sansonetti Philippe J.
Published: October 2006

Shigella flexneri, a Gram‐negative bacillus belonging to the family Enterobacteriaceae, causes bacillary dysentery in humans by invading colonic epithelial cells. Processes by which epithelial cells, which are not professional phagocytes, may limit the spread of the invading microorganisms are poorly understood. This paper shows that IcsA (VirG), a 120kDa bacterial outer membrane protein responsible for intracellular and cell‐to‐cell spread through polymerization of actin, is a major substrate for phosphorylation by cyclic‐dependent protein kinases. Site‐directed mutagenesis of a sequence encoding phosphorylation consensus motif SSRRASS, located at residues 754–760, almost completely abolished the ability of this protein to be phosphorylated by protein kinase A. Such mutants expressed a ‘super Ics’ phenotype, characterized by an increased capacity to spread from cell‐to‐cell during the first three hours of infection in the HeLa cell infection assay. These data suggest that host‐cell phosphorytation of key virulence proteins located on the bacterial surface may represent a significant host defence mechanism during the invasion process.
Rhizobium leguminosarum has two glucosamine syntheses, GImS and NodM, required for nodulation and development of nitrogen‐fixing nodules
Authors: Marie C. , Barny M.‐A. , Downie J. A.
Published: October 2006

The Rhizobium leguminosarum nodM gene product shows strong homology to the Escherichia coli gImS gene product that catalyses the formation of glucosamine 6‐P from fructose 6‐P and glutamine. DNA hybridization with nodM indicated that, in addition to nodM on the symbiotic plasmid, another homologous gene was present elsewhere in the R. leguminosarum genome. A glucosamine‐requiring mutant was isolated and its auxotrophy could be corrected by two different genetic loci. It could grow without glucosamine when the nodM gene on the symbiotic plasmid was induced or if the cloned nodM gene was expressed from a vector promoter. Alternatively, it could be complemented by a second fragment of R. leguminosarum DNA that carries a region homologous to E. coli gImS. Biochemical assays of glucosamine 6‐P formation confirmed that the two R. leguminosarum genes nodM and gimS have interchangeable functions. No nodulation of peas or vetch was observed with a double nodM gImS mutant, and this block occurred at a very early stage since no root‐hair deformation or infection threads were seen. Nodulation and root‐hair deformation did occur with either the nodM or the gImS mutant, showing that the gene products of either of these genes can be involved in the formation of the lipo‐oligosaccharide nodulation signal. However, the gImS mutant formed nodules that had greatly reduced nitrogen fixation. Constitutive expression of nodM restored nitrogen fixation to the gImS mutant. Therefore the reduced nitrogen fixation probably occurs because gImS is absent and nodM is not normally expressed in nodules and, in the absence of glucosamine precursors, normal bacteroid maturation is blocked.
Osmoprotectants and phosphate regulate expression of phospholipase C in Pseudomonas aeruginosa
Authors: Shoriridge Virginia D. , Lazdunski Andrée , Vasil Michael L.
Published: October 2006

Phospholipase C has been increasingly recognized as a significant virulence determinant in the pathogenesis of Gram‐negative and Gram‐positive infections. Pseudomonas aeruginosa carries two, non‐tandem genes encoding phospholipase C (PLC) activity. One PLC (PLC‐H) haemolyses human and sheep erythrocytes while the other is not haemolytic for these kinds of red blood cells. It was previously determined that the synthesis of both PLCs is regulated by inorganic phosphate (Pi), but little else was known regarding the regulation of these potentially important virulence determinants of P. aeruginosa. In this report, data are presented demonstrating that both PLC genes are regulated at the transcriptional level by Pi and by a P. aeruginosa homologue of the positive regulator of genes in the Pi regulon of Escherichia coli, i.e. PhoB. In addition to Pi, it is also shown in this report that the synthesis of both PLC‐H and PLC‐N is induced by compounds which are not only derived from the substrate product of both enzymes, i.e. phosphorylcholine, but are also known osmoprotectants in eukaryotic and prokaryotic cells. The osmoprotective derivatives of phosphorylcholine which induce the synthesis of PLC in P. aeruginosa include choline, glycine betaine, and dimethylglycine, but not sarcosine (monomethylglycine) or glycine. By constructing mutants which are deficient in the production of each separate PLC and in the production of PhoB it was determined that induction of PLC‐H by the osmoprotective compounds is independent of Pi concentration and PhoB, while induction of PLC‐N by these compounds requires Pi‐deficient conditions and PhoB. It was further demonstrated that while an ionic osmolyte, such as NaCl, inhibited production of both enzymes in Pi‐deficient conditions, the presence of osmoprotective compounds, such as choline, restored synthesis of PLC when P. aeruginosa was grown in 0.5 M NaCl. In contrast to the inhibition of PLC production in 0.5 M NaCl in Pi‐deficient conditions, PLC production in an equivalent osmotic pressure generated by the neutral osmolyte sucrose was not inhibited, and choline markediy induced production of PLC in this ionically neutral, high osmotic environment. These observations are especially relevant to the pathogenic potential of PLC in the dehydrating (hence high osmotic) conditions of the lungs of cystic fibrosis patients.
Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer
Authors: Hoyer Lois L. , Hamilton Allison C. , Steenbergen Susan M. , Vimr Eric R.
Published: October 2006

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn 1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy In S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41 300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragills, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure ol the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.
PsiB, an anti‐SOS protein, is transiently expressed by the F sex factor during its transmission to an Escherichia coli K‐12 recipient
Authors: Bagdasarian Mira , Bailone Adriana , Angulo Jaime F. , Scholz Peter , Bagdasarian Michael , Devoret Raymond
Published: October 2006

PsiB, an anti‐SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence‐deduced molecular mass of 15741 daltons. Using an immuno‐assay with anti‐PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6‐5 piasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn 10 outgoing promoter located upstream of psiB. When we inserted a Tn 10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6‐5 PsiB.

We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti‐SOS function. We observed that PsiB protein was transiently expressed by a wild‐type F sex factor during its transmission to an Escherichia coli K‐12 recipient, In an F+× F‐ cross, PsiB concentration increased at least 10‐fold in F‐ recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasm id replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single‐stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single‐stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory pheno‐type.
Conjugative transfer functions of broad‐host‐range plasmid RK2 are coregulated with vegetative replication
Authors: Motallebi‐Veshareh Mostafa , Balzer Dietmar , Lanka Erich , Jagura‐Burdzy Grazyna , Thomas Christopher M.
Published: October 2006

The kilB locus (which is unclonable in the absence of korB) of broad‐host‐range plasmid RK2 (60kb) lies between the trfA operon (co‐ordinates 16.4 to 18.2kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co‐ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBl) of the region analysed results in a Tra‐ phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBi DNA sequence KilBl is predicted to be 34995Da, In line with Mr= 36000 observed by sodium dodecyl sulphate/potyacrylamide gel electrophoresis, and contains a type I ATP‐binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tume‐ faciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI
Studies on the structure and function of the N‐terminal domain of the pneumococcal murein hydrolases
Authors: Sanz J. M. , Díaz E. , García J. L.
Published: October 2006

The structures of the choline‐dependent pneumococcal murein hydrolases, LYTA amtdase and CPL1 Iysozyme, and the choline‐independent CPL7 Iysozyme were analysed by controlled proteolytic digestions. The trypsin cleavage of the CPL1 and CPL7 lysozymes produced two resistant polypeptides, F1 and F7 respectively, corresponding to the N‐terminal domain of the enzymes, whereas the amidase LYTA was completely hydrolysed by the protease. Interestingly, the F1 and F7 fragments showed a low, but significant, choline‐independent lysozyme activity. Choline reduced the rate of proteolytic hydrolysis of choline‐dependent enzymes, suggesting that the C‐terminal choline‐binding domain adopts a more resistant conformation in the presence of the ligand. On the other hand, the regions encoding the N‐terminal domains of the three enzymes have been cloned and expressed in Escherichia coli, showing that these domains adopt an active conformation even in the absence of their C‐terminal domains. The lower activity shown by the catalytic domains when compared with that of the complete enzymes suggests that the acquisition of a substrate‐binding domain represents a noticeable evolutionary advantage for enzymes that interact with polymeric substrates, allowing them to achieve a higher catalytic efficiency. These results strongly reinforce the hypothesis that the pneumococcal murein hydrolases have been originated by fusion of two structural and functional independent domains, and provide new experimental support to the theory of modular evolution of proteins.
The D1 protein of the photosystem II reaction‐centre complex accumulates in the absence of D2: analysis of a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking cytochrome d559
Authors: Shukla Vipula K. , Stanbekova Gulshan E. , Shestakov Sergey V. , Pakrasi Himadri B.
Published: October 2006

The reaction center core of photosystem II, a multi‐protein membrane bound complex, is composed of a heterodimer of two proteins, D1 and D2. A random mutagenesis technique was used to isolate a photosystem II deficient mutant, CP6t16, of the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Nucleotide sequence analysis showed that the primary lesion in CP6t16 is an ochre mutation introducing a translational stop codon in the psbE gene, encoding the α‐subunit of cytochrome b 559, an integral component of the PSII complex. Analysis of the protein composition of CP6t16 thylakoid membranes isolated in the presence of serine protease inhibitors revealed that, in the absence of cytochrome b 559, the D2 protein is also absent. However, the D1 protein is stably incorporated in these membranes, suggesting that the synthesis and integration of D1 are independent of those of D2 and cytochrome 6559.
Overexpression and surface localization of the Chlamydia trachomatis major outer membrane protein in Escherichia coli
Authors: Koehler Jane E. , Birkelund Sven , Stephens Richard S.
Published: October 2006

The Chlamydia trachomatis major 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 in Escherichia coli The MOMP is surface exposed in C. trachomatis and 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 the E. coli surface. 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 rapidly lethal effect on the L2rM0MP E. coli clone. Although no genetic system exists for Chlamydia, development of a stable, inducible E. coli done 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 response.
The influence of ribosome‐binding‐site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo
Authors: Vellanoweth Robert Luis , Rabinowitz Jesse C.
Published: October 2006

A method is described to determine simultaneously the effect of any changes in the ribosome‐binding site (RBS) of mRNA on translational efficiency in Bacillus subtilis and Escherichia 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. Both B. subtilis and E. coli exhibited similar spacing optima of 7–9 nucleotides. However, B. subtilis translated messages with spacings shorter than optimal much less efficiently than E. coli. In both organisms, AUG was the preferred initiation codon by two‐ to threefold. In E coli GUG was slightly better than (JUG while in B. subtilis UUG 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. subtilis v/as also much less able to tolerate secondary structure in the RBS than E. 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 determinants.
The autocatalytic processing of the subtilisin Carlsberg pro‐region is independent of the primary structure of the cleavage site
Authors: Egnell Per , Flock Jan‐Ingmar
Published: October 2006

Subtilisins 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 (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et 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‐region in cis, whereas cleavage by exogenous subtilisin could still occur in 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 around the cleavage site.
Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase
Authors: Bally Marc , Filloux Alain , Akrim Mohammed , Ball Geneviéve , Lazdunski Andree , Tommassen Jan
Published: October 2006

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas 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 three xcp genes has suggested the conservation of this secretion pathway in many Gram‐negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, 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 other xcp genes, designated xcpR to ‐X, are presented. The N termini of four of the encoded Xcp proteins display similarity to the N‐termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in 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 both systems.
Further studies on Pseudomonas aeruginosa LasA: analysis of specificity
Authors: Peters J. E. , Park S. J. , Darzins A. , Freck L. C. , Saulnier J. M. , Wallach J. M. , Galloway D. R.
Published: October 2006

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene 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 protease.
Biochemical characterization of Escherichia coli DNA helicase I
Authors: Dash Pramod K. , Traxler Beth A. , Panicker Mitradas M. , Hackney David D. , Minkley Edwin G.
Published: October 2006

The gene product of F tral is a bifunctional protein which nicks and unwinds the F plasmid during conjugal DNA transfer. Further biochemical characterization of the Tral protein reveals that it has a second, much lower, Km for ATP hydrolysis, in addition to that previously identified. Measurement of the single‐stranded DNA‐stimulated ATPase rate indicates that there is co‐operative interaction between the enzyme monomers for maximal activity. Furthermore, 18O‐exchange experiments indicate that Tral protein hydrolyses ATP with, at most, a low‐level reversal of the hydrolytic step during each turnover.
Many group A streptococcal strains express two different immunoglobulin‐binding proteins, encoded by closely linked genes: characterization of the proteins expressed by four strains of different M‐type
Authors: Stenberg Lars , O'Toole Paul , Lindahl Gunnar
Published: October 2006

Most group A streptococcal strains are able to bind immunoglobulin (lg) in a non‐immune manner, and the majority of these strains bind both IgA and IgG. Using molecular cloning and immunochemical techniques, we have purified and characterized the Ig Fc‐receptors expressed by four such strains. Two of the strains express a novel type of receptor, designated protein Sir, which binds IgA and IgG of all subclasses, and therefore has broader reactivity than any Fc‐receptor previously described. The other two strains express protein Arp, a receptor that binds IgA of both subclasses, and also binds potyclonal IgG weakly. Characterization of the weak IgG‐binding ability of protein Arp shows that it binds only some monoclonal IgG proteins, in particular those of the IgG3 subclass. The four strains studied here were unexpectedly found to also express a second Ig‐receptor, called protein Mrp, encoded by a gene closely linked to the gene for the first receptor. The Mrp protein does not bind IgA, but it binds IgG molecules of the IgG1, IgG2 and tgG4 subclasses, and it also binds fibrinogen. Binding of fibrinogen has been reported to be a characteristic property of streptococcal M proteins, which suggests that the Mrp protein may be an M protein that also binds Ig. Taken together, all available evidence now indicates that most strains of group A streptococci express two different Ig‐binding proteins, encoded by closely linked genes.
Alielic exchange in Pseudomonas aeruginosa using novel ColE1‐type vectors and a family of cassettes containing a portable oriT and the counter‐selectable Bacillus subtilis sacB marker
Authors: Schweizer Herbert P.
Published: October 2006

An improved method for allele replacement in Pseudomonas aeruginosa was developed. The two main ingredients of the method are: (i) novel ColE1‐type cloning vectors derived from pBR322 and pUC19; and (ii) a family of cassettes containing a portable oriT, the sacB gene from Bacillus subtilis as a counter‐selectable marker, and a chloramphenicol‐resistance gene allowing positive selection of both oriT and sacB. Introduction of plasmid‐borne DNA into the chromosome was achieved in several steps. The DNA to be exchanged was first cloned into the new ColE1‐type vectors. After insertion of the oriT and sacB sequences, these plasmid were conjugally transferred into P. aeruginosa and plasmid integrants were selected. Plating on sucrose‐containing medium allowed positive selection for both plasmid excision and curing since Pseudomonas aeruginosa strains containing the sacB gene in single‐ or multiple copy were highly sensitive to 5% sucrose in rich medium. This procedure was successfully used to introduce an agmR mutation into P. aeruginosa wild‐type strain PA01 and should allow the exchange of any DNA segment into any non‐essential regions of the P. aeruginosa chromosome.
SecY variants that interfere with Escherichia coli protein export in the presence of normal secY
Authors: Shimoike Takashi , Akiyama Yoshinori , Baba Tadashi , Taura Tetsuya , Ito Koreaki
Published: October 2006

As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY‐d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose‐binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild‐type cells. The mutation deletes three amino acids that should be located at the interface of cytopiasmic domain 5 and transmembrane segment 9. We also found that some SecY‐PhoA fusion proteins that lacked carboxy‐terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy‐terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the ‘dominant negative’ effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.
Translation initiation in Escherichia coli: sequences within the ribosome‐binding site
Authors: Ringquist Steven , Shinedling Sidney , Barrick Doug , Green Louis , Binkley Jonathan , Stormo Gary D. , Gold Larry
Published: October 2006

The translational roles of the Shine‐Dalgarno sequence, the initiation codon, the space between them, and the second codon have been studied. The Shine Dalgarno sequence UAAGGAGG initiated translation roughly four times more efficiently than did the shorter AAGGA sequence. Each Shine‐Dalgarno sequence required a minimum distance to the initiation codon in order to drive translation; spacing, however, could be rather long. Initiation at AUG was more efficient than at GUG or UUG at each spacing examined; initiation at GUG was only slightly better than UUG. Translation was also affected by residues 3′ to the initiation codon. The second codon can influence the rate of initiation, with the magnitude depending on the initiation codon. The data are consistent with a simple kinetic model in which a variety of rate constants contribute to the process of translation initiation.
Molecular characterization and regulation of the phosphoglycerate kinase gene from Trichoderma viride
Authors: Goldman G. H. , Geremia R. A. , Caplan A. B. , Vila S. B. , Villarroel R. , Van Montagu M. , Herrera‐Estrellal A.
Published: October 2006

We have isolated the phosphoglycerate kinase gene (pgk) of Trichoderma viride and characterized its expression. Comparison of genomic and cDNA clones allowed the correct deduction of the intron boundaries and the 3′‐end cleavage site of this gene. Primer extension analysis showed that transcription initiated at three start points between ‐296 and ‐298bp upstream of the translational start codon. The promoter sequence contained a number of cis‐acting sequences commonly found in eukaryotic promoters. The pgk transcript analysis of T. viride grown on defined carbon sources showed that neither rate nor growth phase greatly affects pgk expression. By contrast, when Trichoderma spp. were grown in the presence of cell walls of a phytopathogenic fungus as carbon source, pgk messenger levels dropped markedly. This suggests that pgk mRNA accumulation is specifically repressed in the simulated mycoparasitic state.
The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N‐terminal repeats
Authors: Coutinho J. B. , Gilkes N. R. , Warren R. A. J. , Kilburn D. G. , Miller R. C.
Published: October 2006

Endoglucanase C (CenC) from Celluiomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150‐amino‐acid repeats (N1 and N2) at its N‐terminus and two unrelated contiguous 100‐amino‐acjd repeats (C1 and C2) at its C‐terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non‐crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated (amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose‐binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose‐binding domains of Cex and C. fimi endoglucanases A and B.
Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic machanism
Authors: Aboagye‐Kwarteng T. , Smith K. , Fairlamb A. H.
Published: October 2006

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.