bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

SummaryThe 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

SummaryExpression 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

SummaryThe 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

SummaryWe 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

SummaryThe 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

SummaryVirtually 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

Summary 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

SummaryThe 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

SummaryPhospholipase 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

SummaryThe 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

SummaryPsiB, 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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryThe 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.