bluuebunny/crossref_metadata_2025 huggingface dataset:
The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci
Authors: Nakata N. , Tobe T. , Fukuda I. , Suzuki T. , Komatsu K. , Yoshikawa M. , Sasakawa C.
Published: October 2006

A large plasmid‐encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin In the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K‐12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K‐12. Conversely, upon introduction of the ompT gene of E. coii K‐12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K‐12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K‐12 and a virulence locus for provoking keratocon‐junctivitis in the eyes of guinea‐pigs, named kcpA is located near purE in S. fiexnerl, and the two loci are involved in VirG expression, the KcpA∼ mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompr gene from E. coli K‐12, giving rise to a KcpA phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.
Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis
Authors: Uchida I. , Makino S. , Sasakawa C. , Yoshikawa M. , Sugimoto C. , Terakado N.
Published: October 2006

Bacillus anthracis produces a gamma‐linked poly‐D‐glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low‐molecular‐weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region atone caused production of a high‐molecular‐weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51460. By in vitro transcription‐translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian γ‐glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly‐D‐glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low‐molecular‐weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.
The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation
Authors: Patriarca E. J. , Riccio A. , Taté R. , Colonna‐Romano S. , Iaccarino M. , Defez R.
Published: October 2006

We report here that ntrB and ntrC genes of Rhizobium leguminosarum biovar phaseoli are cotranscribed with an open reading frame (called 0RF1) of unknown Unction. The promoter region of the 0RF1‐ntrB‐ntrC operon was mapped immediately upstream of ORF1 and two in vivo transcription initiation sites were identified, both preceded by −35/−10 promoter consensus sequences. Some major aspects differentiate ft leguminosarum from the enteric nitrogen regulatory system: the ntrBC genes are cotranscribed with 0RF1 which is homologous to an ORF located upstream of ntrBC of R. capsulatus and to the 0RF1 located upstream of the fis gene of Escherichia coir, ntrBC are not transcribed from a −24/−12 promoter and are only autogenously repressed. Moreover, the intracellular concentration of the NtrC protein increases when the bacterium is grown on ammonium salts, white under the same conditions the promoter of one of its target genes, glnII, is 12 times less active.
Chemotaxis plays a role in the social behaviour of Myxococcus xanthus
Authors: Shi Wenyuan , Köhler Thilo , Zusman David R.
Published: October 2006

Myxococcus xanthus is a Gram‐negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short‐chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl‐accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient‐rich medium. In wild‐type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.
Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae
Authors: Dowson Christopher G. , Coffey Tracey J. , Kell Christopher , Whiley Robert A.
Published: October 2006

Penicillin‐resistant strains of Streptococcus pneumoniae possess forms of penicillin‐binding proteins (PBPs) that have a low affinity for penicillin compared to those from penicillin‐sensitive strains. PBP genes from penicillin‐resistant isolates are very variable and have a mosaic structure composed of blocks of nucleotides that are similar to those found in PBP genes from penicillin‐sensitive isolates and blocks that differ by up to 21%. These chromosomally encoded mosaic genes have presumably arisen following transformation and homologous recombination with PBP genes from a number of closely related species. This study shows that PBP2B genes from many penicillin‐resistant isolates of S. pneumoniae contain blocks of nucleotides originating from Streptococcus mitis. In several instances it would appear that this material alone is sufficient to produce a low affinity PBP2B. In other examples PBP2B genes possess blocks of nucleotides from S. mitis and at least one additional unidentified species. Mosaic structure was aiso found in the PBP2B genes of penicillin‐sensitive isolates of S. mitis or S. pneumoniae. These mosaics did not confer penicillin resistance but nevertheless reveal something of the extent to which localized recombination occurs in these naturally transformable streptococci.
The Hsp60 protein of Helicobacter pylori: structure and immune response in patients with gastroduodenal diseases
Authors: Macchia Giovanni , Massone Annalisa , Burroni Daniela , Covacci Antonello , Censini Stefano , Rappuoli Rino
Published: October 2006

Helicobacter pylori is a human pathogen that has been associated with gastritis, peptic ulcer and gastric carcinoma. The role of the direct action of H. pylori virulence factors and of the induction of autoreactive immunity in the development of chronic gastritis has not been clarified yet. Here we report the cloning and molecular characterization of a gene of H. pylori coding for a protein of 58kDa, recognized by sera of patients affected by H. pylori‐induced gastroduodenal diseases. This antigen is present in all the H. pylori strains tested and it belongs to the Hsp60 family of heat‐shock proteins, with high homology with other bacterial and eukaryotic proteins of the same family. This class of homologous proteins has been implicated in the induction of autoimmune disorders in different systems. The presence in infected patients of anti‐H. pylori Hsp60 antibodies, potentially cross‐reacting with the human homologue, and cross‐reactivity between human Hsp60 and a rabbit antiserum against H. pylori Hsp60 suggest that a role of this protein in gastroduodenal diseases is possible.
The filamentous haemagglutinin, a multifaceted adhesin produced by virulent Bordetella spp.
Authors: Locht Camille , Berlin Philippe , Menozzi Franco D. , Renauld† Genevieve
Published: October 2006

Filamentous haemagglutinin (FHA) is the major attachment factor produced by virulent Bordetella spp. Similar to the other virulence factors, its production is tightly regulated by a two‐component system in response to environmental changes. Although of impressive size (c. 220 kDa), it is very efficiently released into the culture supernatant of Bordetella pertussis. Its biogenesis involves complex processing of a larger precursor with a calculated molecular mass of 370 kDa. Export of FHA into the culture medium depends on an outer membrane protein homologous to haemolysin accessory proteins. Purified extracellular FHA is able to increase the adherence of other pathogens to the host, which may contribute to super‐infection in whooping cough. Although FHA‐ mutants colonize lungs as efficiently as the wild‐type parent strains, immune responses against FHA appear to protect against colonization. Unlike many other adhesins, FHA expresses at least three different attachment activities, one specific for the CR3 integrins of macrophages, one involving a carbohydrate‐binding site, specific for interactions with cilia, and a heparin‐binding activity that may be important for interaction of B. pertussis with epithelial cells or extracellular matrices.
The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility
Authors: Miller Christine A. , Cohen Stanley N.
Published: October 2006

The incompatibitity that pSC101‐derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superheliclty, can function as a cis‐acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101‐specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower‐copy‐number plasmid. We propose a model by which the par locus can enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.
Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III
Authors: Koraimann Günther , Schroller Christa , Graus Hans , Angerer Doris , Teferle Karin , Högenauer Gregor
Published: October 2006

Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19‐lacZ fusion in isogenic RNase III+ and RNase III‐ strains we could identify RNase III as the key element in the down‐regulation of gene 19 expression. The sequencing of in vitro generated and RNase Ill‐digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.
The transcriptional activator HIyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression
Authors: Williams S. G. , Attridge S. R. , Manning P. A.
Published: October 2006

HlyU upregulates expression of the haemolysin, HlyA, of Vibrio cholerae. DNA sequence analysis indicates that HlyU is an 11.9 kDa protein containing a putative helix‐turn‐helix motif and belonging to a family of small regulatory proteins, including NolR (Rhizobium meliloti), SmtB (Synechococcus PCC 7942) and ArsR (piasmids R773, Escherichia coli; p1258, Staphylococcus aureus; and pSX267, Staphylococcus xylosus). An hlyU mutant was constructed by insertional inactivation, and found to be deficient in the production of both the haemolysin and a 28kDa secreted protein. The mutant was assessed for virulence in the infant mouse cholera model, revealing a 100‐fold increase in the LD50.. This suggests that HlyU promotes expression of virulence determinant(s) in vivo.
An unusually small gene required for sporulation by Bacillus subtilis
Authors: Levin Petra Anne , Fan Nancy , Ricca Ezio , Driks Adam , Losick Richard , Cutting Simon
Published: October 2006

We report the cloning and characterization of an unusually small gene called spoVM whose product Is required for normal formation of the cortex and coat during sporulation in Bacillus subtilis. The spoVM gene is adjacent to, and in convergent orientation with, the B. subtilis homologue to the Escherichia coli gene for ribosomal protein L28. The spoVM open reading frame is only 26 codons in length and is capable of encoding a polypeptide of 3 kDa. The short length of spoVM was verified by means of complementation experiments with wild‐type and deletion‐mutated copies of the open reading frame and by engineering the synthesis of the spoVM gene product in E. coli. Transcription of spoVM was induced during the second hour of sporulation (approximately stage II) by the appearance of the sporulation RNA poly‐merase sigma factor, σ;E. Efficient transcription of spoVM additionally required the action of the sporulation DNA‐binding protein SpolllD. Because spoVM was not strongly required for the transcription of several genes expressed at late times in development, its protein product is likely to play a morphogenetic rather than a regulatory role in sporulation.
Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence
Authors: Bassler Bonnie L. , Wright Miriam , Showalter Richard E. , Silverman Michael R.
Published: October 2006

Density‐dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two–component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal–response system controlling density‐dependent expression of luminescence remains to be identified.
Suppression of a dominant G protein β‐subunit mutation in yeast by Gα protein expression
Authors: Zhang Meng , Tipper Donald J.
Published: October 2006

SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in mating pheromone signal transduction in Saccharomyces cerevisiae. Responses, including G1 arrest and expression of genes such as FUS1, are activated by βλ, which is negatively controlled by α(GDP), We previously demonstrated that overexpression of Scg1 suppresses responses to α factor and that expression of certain hybrids between Scg1 and mammalian Gα proteins has the same effect and also suppresses growth arrest in an scg1‐null mutant. Effects were attributed to sequestration of βλ. We now show that effects on growth rate, morphology and FUS1 expression are consistent with this model. The STE4HPL allele causes dominant activation of the response pathway, and is presumed to encode a β subunit insensitive to control by α(GDP). Scg1 overexpression suppresses the growth arrest due to STE4HPL; normal α‐factor responses and fertility are restored. A model based on sequestration of βγ reconciles this result with the apparent paradox that the same level of Scg1 overexpression inhibits responses and mating in wild‐type cells. A Gαi hybrid also restores growth and allows inefficient mating in the STEHPL strain.
Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene, dcm
Authors: Dar Mubasher E. , Bhagwat Ashok S.
Published: October 2006

The DNA cytosine methylase gene of Escherichia coli, dem, overlaps an open reading frame (ORF) that continues in +1 register past the end of dcm. This ORF codes for a gene, vsr, that is required for a T:G to C:G base mismatch correction process. In this study, mutants that affect the level of expression of the two genes were constructed and characterized. Further, a previously isolated mutant, dcm‐6, was cloned and mutations within it were identified. Northern blots were used to identify dcm‐specific RNA species in wild type and dcm‐6 cells. Based on these studies we conclude that there is a six‐codon overlap between vsr and dcm. The two proteins appear to be made from a single RNA transcript and translation of dcm is required for the efficient synthesis of Vsr. Further, Vsr is active by itself and may not be produced as a fusion with Dem. This is the first example of chromosomal genes that overlap in their coding regions and produce proteins with distinct functions.
Isozymes of S‐adenosylmethionine synthetase are encoded by tandemly duplicated genes in Escherichia coli
Authors: Satishchandran C. , Taylor John C. , Markham George D.
Published: October 2006

The sole biosynthetic route to S‐adenosylmethionine, the primary biological alkylating agent, is catalysed by S‐adenosylmethionine synthetase (ATP: L‐methionine S‐adenosyltransferase). In Escherichia coli and Sal‐monella typhimunum numerous studies have located a structural gene (metK) for this enzyme at 63min on the chromosomal map. We have now identified a second structural gene for S‐adenosylmethionine synthetase in E. coli by DNA hybridization experiments with metK as the probe; we denote this gene as metX. The metX gene is located adjacent to metK with the gene order speA metK metX speC. The metK and metX genes are separated by ∼0.8kb. The metK and the metX genes are oriented convergently as indicated by DNA hybridization experiments using sequences from the 5′ and 3′ ends of metK. The metK gene product is detected immunochemically only in cells growing in minimal media, whereas the metX gene product is detected immunochemically in cells grown in rich media at all growth phases and in stationary phase in minimal media.

Mutants in metK or metX were obtained by insertion of a kanamycin resistance element into the coding region of the cloned metK gene (metK:: kan), followed by use of homologous recombination to disrupt the chromosomal metK or metX gene. The metK::kan mutant thus prepared does not grow on minimal media but does grow normally on rich media, while the corresponding metX::kan mutant does not grow on rich media although it grows normally on minimal media. These results indicate that metK expression is essential for growth of E. coli on minimal media and metX expression is essential for growth on rich media. Our results demonstrate that Ado Met synthetase has an essential cellular and/or metabolic function. Furthermore, the growth phenotypes, as well as immunochemical studies, demonstrate that the two genes that encode S‐adenosylmethionine synthetase isozymes are differentially regulated. The mutations in metK and metX are highly unstable and readily yield kanamycin‐resistant cells in which the chromosomal location of the kanamycin‐resistance element has changed.
Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa
Authors: Martin Paul R. , Hobbs Matthew , Free Patricia D. , Jeske Yvette , Mattick John S.
Published: October 2006

Type 4 fimbriae are produced by a variety of pathogens, in which they appear to function in adhesion to epithelial cells, and in a form of surface translocation called twitching motility. Using transposon mutagenesis of Pseudomonas aeruginosa, we have identified a new locus required for fimbrial assembly. This locus contains the gene pilQ which encodes a 77 kDa protein with an N‐terminal hydro‐phobic signal sequence characteristic of secretory proteins, pilQ mutants lack the spreading colony morphology characteristic of twitching motility, are devoid of fimbriae, and are resistant to the fimbrial‐specific bacteriophage PO4. The pilQ gene was mapped to Spel fragment 2, which is located at 0–5 minutes on the P. aeruginosa PAO1 chromosome, and thus it is not closely linked to the previously characterized pilA‐D, pilS,R or pilT genes. The pilQ region also contains ponA, aroK and aroB‐like genes in an organization very similar to that of corresponding genes in Escherichia coli and Haemophilus influenzae. The predicted amino acid sequence of PilQ shows homology to the PulD protein of Kleb‐siella oxytoca and related outer membrane proteins which have been found in association with diverse functions in other species including protein secretion, DNA uptake and assembly of filamentous phage. PilQ had the highest overall homology to an outer membrane antigen from Neisseria gonorrhoeae, encoded by omc, that may fulfil the same role in type 4 fimbrial assembly in this species.
Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii
Authors: Blanco Gonzalo , Drummond Martin , Woodley Paul , Kennedy Christina
Published: October 2006

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C‐terminal domain to the histidine protein kinases (HPKs) than Is K. pneumoniae NifL. In particular A. vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems. Both NifL proteins are homologous in their N‐termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio‐opsin, the expression of which is oxygen sensitive. The same region showed homology to the haembinding N‐terminai domain of the Rhizobium meliloti fixL gene product, an oxygen‐sensing protein. Like K. pneumoniae NifL, A. vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH+4 or oxygen. The sequences found homologous in the C‐terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing. An in‐frame deletion mutation in the nifL coding region resulted in loss of repression by NH+4 and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, nifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expression from the A. vinelandii promoter requires neither RpoN nor NtrC.
Molecular characterization of a fungal secondary metabolism promoter: transcription of the Aspergillus nidulans isopenicillin N synthetase gene is modulated by upstream negative elements
Authors: Perez‐Esteban Beatriz , Orejas Margarita , Gómez‐Pardo Emilia , Peñalva Miguel Angel
Published: October 2006

The Aspergillus nidulans IPNS gene, encoding isopenicillin N synthetase, is a secondary metabolism gene. It is contiguous to, but divergently transcribed from, the ACVS gene at the penicillin gene cluster. The untranslated region between both ORFs is 872bp long. Here we present the physical and functional characterization of the IPNS transcriptional unit. Transcriptional start point (tsp) mapping reveals heterogeneity at the 5′‐end of the mRNA, with a major start at −106 relative to the initiation codon. This indicates that the actual length of the non‐transcribed intergenic region is 525bp. Functional elements in the IPNS upstream region have been defined by assaying β‐galactosidase activity in extracts from recombinant strains carrying deletion derivatives of the IPNS promoter fused to lacZ, integrated in single copy at the argB locus. Strains were grown in penicillin production broth under carbon catabolite repressing or derepressing conditions. The results of deletion analysis indicate that: (i) the IPNS promoter is mostly regulated by negative controls that act upon a high basal activity; (ii) sequential deletion of three of the negative cis‐acting elements results in a mutated promoter that is 40 times (sucrose broth) or 12 times (lactose broth) more active than the wild type; (iii) one of these negative cis‐acting elements is involved in sucrose repression. Strikingly, it is located outside the non‐transcribed 525 bp intergenic region and maps to the coding region of the divergently transcribed ACVS gene; (iv) a 5′‐del‐etion up to −56 (relative to the major tsp) contains information to provide almost half of the maximal promoter activity and allows initiation of transcription at the correct site. By using total‐protein extracts from mycelia grown under penicillin producing conditions we have detected a DNA‐binding activity that specifically shifts a promoter fragment located between −654 and −455(relative to IPNS tsp). Deletions covering this region partially abolish IPNS promoter activity. The fragment in question overlaps the ACVS tsp.
Gene‐specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2
Authors: Hinnebusch Alan G.
Published: October 2006

Phosphorylation of the α subunit of eukaryotic initiation factor 2 (elF‐2α) is one of the best‐characterized mechanisms for down‐regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene‐specific case of translational control by phosphorylation of elF‐2α, Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORFI fail to reinitiate at uORFs 2‐4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2‐4 in starved cells results from a reduction in the GTP‐bound form of elF‐2 that delivers charged initiator tRNAiMet to the ribosome. When the levels of elF‐2·GTP·Met‐tRNAiMet ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of elF‐2 by the protein kinase GCN2 decreases the concentration of elF‐2·GTP·Met‐tRNAiMet complexes by inhibiting the guanine nucleotide exchange factor for elF‐2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of elF‐2.
Control and function of lysyl‐tRNA synthetases: diversity and co‐ordination
Authors: Nakamura Yoshikazu , Ito Koichi
Published: October 2006

Lysyl‐tRNA synthetases are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly); however, the physiological significance and the differential control mechanism of these two genes have been a long‐standing puzzle. Recent studies have successfully uncovered a significant control mechanism of lysU expression, which involves the leucine‐responsive regulatory protein (Lrp) and a translational enhancer element called‘downstream box'. Moreover, it is likely that there is a mechanism underlying co‐ordinate expression of lysU with other genes outside the leucine‐Lrp regulon under harsh conditions such as low pH and anaerobiosis. A possible mechanism of lysyl‐tRNA synthetase expression and function is reviewed.
Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein‐secretion apparatus: a general system for the formation of surface‐associated protein complexes
Authors: Hobbs Matthew , Mattick John S.
Published: October 2006

The Pseudomonas aeruginosa genes pilB‐D and pilQ are necessary for the assembly of type 4 fimbriae. Homologues of these genes and of the subunit (pilin) gene have been described in various different bacterial species, but not always in association with type 4 fimbrial biosynthesis and function. Pil‐like proteins are also involved in protein secretion, DNA transfer by conjugation and transformation, and morphogenesis of filamentous bacteriophages. It seems likely that the Pil homologues function in the processing and export of proteins resembling type 4 fimbrial sub‐units, and in their organization into fimbrial‐like structures. These may either be true type 4 fimbriae, or components of protein complexes which act in the transport of macromolecules (DNA or protein) into or out of the cell. Some PilB‐like and PilQ‐like proteins are apparently also involved in the assembly of non‐type 4 polymeric structures (filamentous phage virions and conjugative pili). The diverse studies summarized in this review are providing insight into an extensive infrastructural system which appears to be utilized in the formation of a variety of cell surface‐associated complexes.
The galactose regulon of Escherichia coli
Authors: Weickert Michael J. , Adhya Sankar
Published: October 2006

Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by two different galactose‐specific transport systems. At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences. These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The negative regulators, GalR and GalS, discriminate between utilization of the high‐affinity (regulated by GalS) and low‐affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP. Both promoters are strongly repressed by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have two operators. The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.
Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis
Authors: Grundy Frank J. , Waters Debra A. , Takova Tsetska Y. , Henkin Tina M.
Published: October 2006

The Bacillus subtilis ccpA gene has previously been shown to be involved in repression of amyE expression when cells are grown in excess glucose. The region of the B. subtilis chromosome downstream from ccpA was characterized to determine if additional genes involved in carbohydrate metabolism were present. Two open reading frames that exhibited sequence similarity to the Escherichia coli and B. subtilis motA and motB motility genes were found immediately downstream from ccpA; disruption of this region had no effect on growth, sporulation or motility. Two divergent transcriptional units containing the acsA and acuABC genes were also found in this region. The acsA gene encodes acetyl‐CoA synthetase, and inactivation of this gene resulted in loss of the ability to utilize acetate as a carbon source for growth or sporulation. Disruption of the acuABC genes resulted in poor growth or sporulation on acetoin or butanediol. The acsA and acuABC promoter sequences were identified by primer extension, and are in close proximity. Two sequences resembling the amyO regulatory target site necessary for glucose repression of amyE were identified in the acsA‐acuABC promoter regions.
Linker‐insertion mutagenesis of Pseudomonas aeruginosa outer membrane protein OprF
Authors: Wong Rebecca S. Y. , Jost Helen , Hancock Robert E. W.
Published: October 2006

The oprF gene, expressing Pseudomonas aeruginosa major outer membrane protein OprF, was subjected to semi‐random linker mutagenesis by insertion of a 1.3 kb Hincll kanamycin‐resistance fragment from plasmid pUC4KAPA into multiple blunt‐ended restriction sites in the oprF gene. The kanamycin‐resistance gene was then removed by Pstl digestion, which left a 12 nucleotide pair linker residue. Nine unique clones were identified that contained such linkers at different locations within the oprF gene and were permissive for the production of full‐length OprF variants. In addition, one permissive site‐directed insertion, one non‐permissive insertion and one carboxy‐terminal insertion leading to proteolytic truncation were also identified. These mutants were characterized by DNA sequencing and reactivity of the OprF variants with a bank of 10 OprF‐specific monoclonal antibodies. Permissive clones produced OprF variants that were shown to be reactive with the majority of these monoclonal antibodies, except where the insertion was suspected of interrupting the epitope for the specific monoclonal antibody. In addition, these variants were shown to be 2‐mercaptoethanol modifiable, to be resistant to trypsin cleavage in intact cells and partly cleaved to a high‐molecular‐weight core fragment in outer membranes and, where studied, to be accessible to indirect immunofluorescenee labelling in intact cells by monoclonal antibodies specific for surface epitopes. Based on these data, a revised structural model for OprF is proposed.
The ferric iron‐binding protein of pathogenic Neisseria spp. functions as a periplasmic transport protein in iron acquisition from human transferrin
Authors: Chen Cheng‐Yen , Berish Sally A. , Morse Stephen A. , Mietzner Timothy A.
Published: October 2006

The ferric iron‐binding protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high‐affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram‐negative periplasmic‐binding protein component, which operates as a part of a general active transport process for the receptor‐mediated, high‐affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well‐characterized periplasmic‐binding proteins, including structural features and the reversible binding of ligand. Predictive of a periplasmic‐binding protein, which functions in the high‐affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse–chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a periplasmic‐binding protein‐mediated active transport mechanism for the acquisition of iron from human transferrin.
Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae
Authors: Loubens I. , Debarbieux L. , Bohin A. , Lacroix J.‐M. , Bohin J.‐P.
Published: October 2006

Membrane‐derived oligosaccharides (MDO) of Escherichia coli are representative members of a family of glucans found in the periplasmic space of Gram‐negative bacteria. The two genes forming the mdoGH operon are necessary for the synthesis of MDO. The nucleotide sequence (4759 bp) and the transcription‐al start of this operon were determined. Both gene products were further characterized by gene fusion analysis. MdoG is a 56 kDa periplasmic protein whose function remains to be determined. MdoH, whose presence was shown to be necessary for normal glucosyl transferase activity, is a 97 kDa protein spanning the cytoplasmic membrane. To our surprise, these proteins are not homologous to the periplasmic glucan biosynthetic enzymes previously characterized in the Rhizobiaceae family. However, a considerable homology (69% identical nucleotides out of 2816) was discovered between mdoGH and the two genes present at the hrpM locus of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Functions of these genes remain mysterious but they are known to be required for both the expression of disease symptoms on host plants and the development of the hypersensitive reaction on non‐host plants (Mills and Mukhopadhyay, 1990). These results confirm the importance of periplasmic glucans for the physiological ecology of Gram‐negative bacteria.
A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli
Authors: Ishizuka Hitoshi , Hanamura Akemi , Kunimura Tadashi , Aiba Hiroji
Published: October 2006

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and β‐galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP‐overproducing cells, while it does not fully reverse the effect of glucose on β‐galactosidase expression in wild‐type cells. When the CRP concentration is reduced by manipulating the crp gene, β‐galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.
Resistance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an O‐acetylation of the Rhizobium leguminosarum lipo‐oligosaccharide nodulation factor
Authors: Firmin J. L. , Wilson K. E. , Carlson R. W. , Davies A. E. , Downie J. A.
Published: October 2006

Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv. Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R. I. viciae. One strain (TOM) which can nodulate cv. Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance. Strain TOM makes significantly lower amounts of lipo‐oligosaccharide nodulation factors than other strains of R. I. viciae. and this effect appears to be due to lower levels of nod gene induction. These nodulation factors are similar to those from other R. I. viciae. strains in that they consist of an oligomer of four or five β1‐4‐linked N‐acetylglucosamine residues in which the terminal non‐reducing glucosamine carries an O‐acetyl group and a C18:4 or C18:1N‐acyl group. However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R. I. viciae. in that it carries an O‐acetyl group on the C‐6 of the reducing N‐acetylglucosamine residue. This acetylation is NodX‐dependent and the pentameric nodulation factor is acetylated on the reducing N‐acetylglucosamine residue whereas the tetrameric nodulation factor is not. Although the nodL gene product is also an O‐acetyl transferase (it O‐acetylates the C‐6 of the terminal non‐reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.
Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis
Authors: Jennings Michael P. , van der Ley Peter , Wilks Kathy E. , Maskell Duncan J. , Poolman Jan T. , Moxon E. Richard
Published: October 2006

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE‐coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.
Regulatory implications of translational frameshifting in cellular gene expression
Authors: Engelberg‐Kulka Hanna , Schoulaker‐Schwarz Rachel
Published: October 2006

The genetic code, once thought to be rigid, hag been found to be quite fiexible, permitting several different reading alternatives. One of these is translatlonal frameshifting, a process programmed in the mRNA sequence and which enables a +1 or ‐1 shift from the reading frame of the initiation codon. So far, the Involvement of translatlonal frameahifting in gene expression has been described mainly in viruses (particularly retroviruses), retrotransposons, and bacterial insertion elements, in this MicroReview., we present a survey of the cellular genes, mostly in Escherichia coil, which have been found to be expressed through a transiational frameshifting process, as well as a discussion of the regulatory implications of this process.
Mammalian and Escherichia coli signal recognition particles
Authors: Luirink Joen , Dobberstein Bernhard
Published: October 2006

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the pro‐karyotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleo‐protein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses these recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.
Clonal spread of serogroup A meningococci: a paradigm for the analysis of microevolution in bacteria
Authors: Achtman Mark
Published: October 2006

Extensive epidemiological analyses of epidemics of meningococcal meningitis have resulted in large, well‐defined strain collections which represent the local diversity and global spread of serogroup A bacteria. Several genes for cell surface proteins are conserved during spread, with a few exceptions: analysis of these exceptions has revealed some of the phenomena which can lead to microevolution. Micro‐evolution is so rapid with serogroup A meningococcal that several independent recombination events have been documented within the last few decades. In a few cases, the recombinant bacteria have become established by clonal replacement plus epidemic spread. Comparison with other bacteria indicates that serogroup A meningococcal provide a number of advantages for analysis of microevolution.
Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri
Authors: Suzuki T. , Murai T. , Fukuda I. , Tobe T. , Yoshikawa M. , Sasakawa C.
Published: October 2006

Intercellular spreading of shigellae Is a prerequisite for shigellosis, although the molecular mechanisms underlying the phenomenon are still largely obscure. To elucidate some of these mechanisms, we performed random TniO insertion mutagenesis in Shigella flexneri YSH6000T and found a chromosomal locus in the Notl‐J segment responsible for bacterial spreading. The locus affected in the mutant, designated vacJ, was neither involved in the invasion of epithelial cells nor in intracellular movement, but was required for intercellular spread. The vacJ mutant was capable of forming bacterium‐containing membranous protrusions within the infected cell, but had diminished ability to move from the protrusions into the cytoplasm of the adjacent epithelial cells. Cloning and sequencing of the vacJ region Indicated that the vacJ gene encoded a 28.0 kDa protein possessing a signal peptide at the N‐terminus, which contained the motif characteristic of lipoproteins. The analysis of the vacJ product indicated that VacJ was exposed on the bacterial surface. The vacJ gene was distributed among shigellae and enteroinvasive Escherichia coli, and the constructed vacJ mutants failed to spread intercellularly, indicating that vacJ is a chromosomal gene essential for the pathogenicity of shigeiiae.