bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing
Authors: Nilsson P. , Uhtin B. E. 
Published: October 2006

SummaryDifferential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post‐transcriptional processing event, a papA gene‐specific mRNA product (mRNA‐A) accumulates in large excess relative to the primary mRNA‐BA transcript. Our results show that the processed product, mRNA‐A, is a translationally active molecule and that it is generated from the mRNA‐BA precursor by an RNaseE‐dependent mechanism. The processing did not occur under non‐permissive conditions in an E. coli me mutant strain with a temperature‐sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA‐BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaselll and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/alletic to rne. Absence of the processing event in the RNaseE mutant (me‐3071) strain led to a four‐fold stabilization of the mRNA‐BA primary transcript. We conclude that the RNaseE‐dependent processing event is the rate‐limiting step in the decay of the papB‐coding part of the primary transcript and in the production of the stable mRNA‐A product. The present results with the papBA transcripts and previous analyses of rne mutants clearly suggest an important role for RNaseE in turnover of several E. coli mRNAs. Furthermore, the novel processing event exemplified by the pap system demonstrates that RNaseE can have a crucial role at the post‐transcriptional stage in the production of stable and efficiently translated mRNA molecules from E. coli operons.
The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes
Authors: Bender R. A. 
Published: October 2006

SummaryThe NAC (nitrogen assimilation control) protein from Klebsiella aerogenes is a LysR‐like regulator for transcription of several operons involved in nitrogen metabolism, and couples the transcription of these σ70‐dependent operons to regulation by the σ54‐dependent NTR system. NAC activates expression of operons (e.g. histidine utilization, hut), allowing use of poor nitrogen sources, and represses expression of operons (e.g. glutamate dehydrogenase, gdh) allowing assimilation of the preferred nitrogen source, ammonium. NAC is both necessary and sufficient to activate transcription, but the expression of the nac gene is totally dependent on the central nitrogen regulatory system (NTR) and RNA polymerase carrying the σ54 sigma factor (RNAPσ54). Nitrogen starvation signals the NTR system to transcribe nac, and NAC activates the transcription of hut, put (pro‐line utilization), and urease. NAC does not affect the transcription of RNAP σ54‐dependent operons like gInA or nifLA, which respond directly to the NTR system, but activates transcription of RNAPσ70‐dependent operons. Thus NAC acts as a bridge between RNAPσ70‐dependent operons like hut and the RNAPσ54‐dependent NTR system. The activation of operons like hut by NAC in response to nitrogen starvation is at least superficially similar to their activation by CAP‐cAMP in response to carbon and energy starvation.
Pseudomonad replication origins: a paradigm for bacterial origins?
Authors: Smith D. W. , Yee T. W. , Baird C. , Krishnapiilai V. 
Published: October 2006

SummaryStructural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compared in order to deduce characteristics common to all bacterial origins and characteristics that distinguish the three origin classes. The two Pseudomonas aeruginosa origins are shown to map within 10 kb of each other, and correlations are drawn with four potential origin regions in B. subtilis. The enteric origin class is further distinguished from the other two classes by its genetic organization, the presence of GATC sites, and the role of Dam methylation in enteric initiation. The pseudomonad origin class has the most features that are common to all of these bacterial origins, and hence may be the paradigm bacterial origin class.
Structure and function of DnaA and the DnaA‐box in eubacteria: evolutionary relationships of bacterial replication origins
Authors: Yoshikawa H. , Ogasawara N. 
Published: October 2006

SummaryDnaA protein (a trans‐acting element) and its binding sequence, DnaA‐box: (a cis‐acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram‐positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram‐negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA‐box (DnaA‐box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA‐box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA‐box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.
The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes
Authors: Inoue C. , Sugawara K. , Kusano T. 
Published: October 2006

SummaryTwo distinct merR genes, which regulate expression of the mercuric ion resistance gene (mer), of Thiobacillus ferrooxidans strain E‐15 have been cloned, sequenced and termed merR1, and merR2. As a result of gene walking around two merR genes, it was found that these two genes were quite close in distance. The nucleotide sequence of the region (5001 base pairs; PstI‐EcoRI fragment) containing the merR genes was determined. Between the two merR genes, there were five potential open reading frames (ORFs). Two of these were identified as merC genes, and the other three as ORFs 1 to 3. ORFs 1 to 3 show significant homology to merA, tnsA from transposon Tn7, and merA, respectively. Both merR genes consist of a 408 bp ORF coding for 135 amino acids. Their gene products, MerRl and MerR2, differed at three amino acid positions, and shared 56‐57% and 32‐38% identity with the MerRs from other Gram‐negative and Gram‐positive bacteria, respectively. Competitive primer extension analysis revealed that both regulatory genes were expressed in the host cells. These merR genes were located more than 6 kb from either end of the mer structural genes (merC‐merA). This is the first example of merR being separated from the mer structural genes. The two merC genes, each of which coded for a 140‐amino‐acid protein, appeared to be functionally active because Escherichia coli cells carrying these merC geneson plasmid vectors showed hypersensitivity to HgCl2. However, ORFs 1 and 3, which were homologous to merA, seemed to be inactive both structurally and enzymatically. The gene arrangement in this region took on a mirror image, with the truncated tnsA as the symmetrical centre. It is suggested that the Tn7‐like factor may have participated in gene duplication events of the mer region, and in its chromosomal integration.
Investigation of the specificity of the interaction between colicin E9 and its immunity protein by site‐directed mutagenesis
Authors: Curtis M. D. , James R. 
Published: October 2006

SummaryComparison of the amino acid sequences of the C‐terminal domain of three DNAase type E colicins has identified six candidate specificity determinants for the interaction of these E colicins with their homologous immunity proteins. We have changed these candidate specificity determinants of colicin E9, using site‐directed mutagenesis, to the corresponding amino‐acids of colicin E8. A‘mutant’colicin E9, in which four of the six candidate specificity determininants have been changed, demonstrated colicin activity against Escherichia coli indicator strains which carried either the E8imm or the E9imm genes, indicative of a‘novel’E colicin. After changing all six of the candidate specificity determinants, the resulting colicin E9‘mutant’exhibited a phenotype very similar to that of colicin E8.
Skp is a periplasmic Escherichia coli protein requiring SecA and SecY for export
Authors: Thome B. M. , Müller M. 
Published: October 2006

SummarySkp of Escherichia coli (OmpH of Salmonella typhimurium) is a protein whose precise function has been obscured by its ubiquity in a wide range of sub‐cellular fractions such as those containing DNA, ribosomes, and outer membranes. Combining in vitro and in vivo techniques we show that Skp is synthesized as a larger precursor that is processed upon translocation across the plasma membrane. Translocation is dependent on the H+‐gradient, ATP, SecA, and SecY. Upon cellular subfractionation (avoiding non‐specific electrostatic interactions) Skp partitions with β‐lactamase into the fraction of soluble, periplasmic proteins. In the context of the export factor properties of Skp previously demonstrated in vitro it is conceivable that this protein is involved in the later steps of protein translocation across the plasma membrane and/or sorting to the outer membrane.
A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants
Authors: Torres L. , Martín H. , García‐Saez M. I. , Arroyo J. , Molina M. , Sánchez M. , Nombela C. 
Published: October 2006

SummaryBy genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation. A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484‐amino‐acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle. Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol. However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LVT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V. The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell‐wall functions.
Yeast genes involved in DNA‐repair processes: new looks on old faces
Authors: Friedberg E. C. 
Published: October 2006

SummaryMultiple genes (many of which are designated RAD (for radiation resistance)) are required for cellular responses to DNA damage in the yeast Saccharomyces cerevisiae. In recent years a number of these genes have been cloned and sequenced and in some cases their polypeptide products have been purified and characterized biochemically. These studies are beginning to yield clues about the possible nature of the multiple biochemical pathways for DNA‐damage processing in yeast.
Yeast dsRNA viruses: replication and killer phenotypes
Authors: Tipper D. J. , Schmitt M. J. 
Published: October 2006

SummaryThe cytoplasmic L‐A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5kb dsRNA and the two gene products it encodes: the capsid (cap) and at least one copy of the capsid–polymerase (cap–pol) fusion protein. Virion cap–pol catalyses transcription of the plus (sense)‐strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap–pol. Nascent cap–pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus‐strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L‐A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self‐selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross‐immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell‐wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.
Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae
Authors: Latterich M. , Watson M. D. 
Published: October 2006

SummaryThe yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and α‐mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt‐sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.
Characterization, localization and transmembrane organization of the three proteins PrtD, PrtE and PrtF necessary for protease secretion by the Gram‐negative bacterium Erwinia chrysanthemi
Authors: Delepelaire P. , Wandersman C. 
Published: October 2006

Summary Erwinia chrysanthemi, a Gram‐negative phythopathogenic bacterium, secretes two related extracellular metalloproteases, B and C, which do not have N‐terminal signal sequences. The specific pathway by which they are secreted, which has been reconstituted in Escherichia coli, comprises three proteins — PrtD, PrtE and PrtF. Hybrid proteins containing segments of these proteins fused to the C‐terminus of protease B were purified and used to immunize rabbits. The antisera thus obtained were used to study the location and membrane topology of the three proteins. PrtD and PrtE were found to cofractionate almost exclusively with the cytoplasmic membrane, whereas PrtF was found to co‐fractionate mostly with the outer membrane. Proteinase K accessibility experiments as well as sequence data lead us to propose that PrtF has one or both ends exposed to the periplasm, that PrtE has one transmembrane segment with its amino‐terminus facing the cytoplasm and its C‐terminal hydrophilic domain exposed to the periplasm, and that PrtD has six transmembrane segments with its N‐terminus and its C‐terminal hydrophilic domain in the cytoplasm.
Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG‐like family of proteins
Authors: Venkatesan M. M. , Buysse J. M. , Hartman A. B. 
Published: October 2006

SummaryOligonucleotide primers derived from the ipaH7.8 sequence have been used to determine the boundaries of DNA sequence homology among five lpaH genes on the invasion plasmid (pWR100) of Shigella flexneri 5, strain M9OT‐W. The primary structure of lpaH4.5 has been established from DNA sequence analysis. The first 197 amino acids in lpaH7.8 were replaced in lpaH4.5 by a unique set of 251 amino acids, generating two related proteins with variable and conserved sequences. The amino‐terminal region of lpaH4.5 displayed an internal repeat structure, also seen in lpaH7.8, characteristic of members of the leucine‐rich glycoprotein (LRG) family. The DNA sequences of ipaH2.5 and ipaH1.4 indicate that these genes are truncated versions of lpaH7.8. Western blot analysis of a λgt11 ipaH recombinant (W7) subclone demonstrated that the antigenicity of lpaH7.8 resides outside the leucine‐rich repetitive region.
Physical and genetic map of the Neisseria gonorrhoeae strain MS11‐N198 chromosome
Authors: Bihlmater A. , Römling U. , Meyer T. F. , Tümmler B. , Gibbs C. P. 
Published: October 2006

SummaryA macro‐restriction map of the Neisseria gonorrhoeae chromosome was constructed using the enzymes Nhel and Spel. Combinations of one‐and two‐dimensional electrophoresis of completely or partially digested chromosomal DNA were performed to align the restriction fragments. The chromosome is circular, with an estimated size of 2.33Mb±35kb. A genetic map was derived from the physical map; positions of over 60 defined loci were determined by Southern hybridization.
Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β‐galactosidase fused to the Hly C‐terminal signal domain
Authors: Kenny B. , Haigh R. , Holland I. B. 
Published: October 2006

SummarySecretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post‐translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C‐terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids.In this paper, we have sought to confirm the presence of a C‐terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C‐terminal fragments of HlyA to heterologous polypeptides.A C‐terminal fragment (23 kDa) of HlyA, when fused at the C‐terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloram‐phenicol acetyltransferase (CAT) and 98 per cent of the β‐galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences.Analysis of the nature of the C‐terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.
Molecular aspects of microbial ice nucleation
Authors: Warren G. , Wolber P. 
Published: October 2006

SummaryCertain organisms nucleate the crystallization of ice. This requires a small volume of water to be induced, probably by lattice‐matching with a solid template, to form an‘ice embryo’—a region sharing at least some of the characteristics of macroscopic ice. It is of particular interest to understand the structure and function of biological structures capable of lattice‐matching (or otherwise inducing a quasi‐crystalline state). Some strains of the Gram‐negative eubacterial genera Erwinia, Pseudomonas, and Xanthomonas, and the mycobionts of certain lichens, display ice‐nucleating activity. In bacteria, the activity is conferred by a protein that contains three nested periodicities of repetition, which probably reflects a hierarchy of three motifs of structural repetition. Thus the tertiary structure of the ice‐nucleation protein is likely to be regular, consistent with the expectation of its forming a template for lattice‐matching. Even within a clonal culture, the nucleating sites formed by bacteria and lichens vary considerably in the threshold temperatures at which they display activity; this indicates wide variations in either the size of the template, or its structural regularity, or both. However, ice‐nucleating sites of lichen and bacterial origin are clearly differentiated by their sensitivities to experimental treatments.
The stringent response in Streptomyces coelicolor A3(2)
Authors: Strauch E. , Takano E. , Baylts H. A. , Bibb M. J. 
Published: October 2006

SummaryThe stringent response was elicited in the antibiotic producer Streptomyces coelicolor A3(2) either by amino acid depletion (nutritional shiftdown) or by the addition of serine hydroxamate; both led to increased levels of ppGpp and to a reduction in transcription from the four promoters of the rrnD rRNA gene set. Analysis of untreated batch cultures revealed elevated ppGpp levels at the end of exponential growth, preceding the onset of antibiotic production. The effect of provoking the stringent response on antibiotic production in exponentially growing cultures was assessed by S1 nuclease mapping of actIII, an early gene of the actinorhodin biosynthetic cluster. Expression of act III occurred after nutritional shiftdown, but not after treatment with serine hydroxamate. Although the need for ppGpp in triggering antibiotic production remains equivocal, ppGpp synthesis atone does not appear to be sufficient to initiate secondary metabolism in S. coelicolor A3(2).
Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b
Authors: Cardy D. L. N. , Laidler V. , Salmond G. P. C. , Murrell J. C. 
Published: October 2006

SummaryThe oxidation of methane to methanol in methano‐trophic bacteria is catalysed by the enzyme methane monooxygenase (MMO). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the α, β and γ subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.