bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiology and molecular genetics of poly(β‐hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus
Authors: Steinbüchel A. , Schlegel H. G.
Published: October 2006

The Alcaligenes eutrophus genes for β‐ketothiolase, NADPH‐dependent acetoacetyl‐CoA reductase and poly(β‐hydroxybutyric acid) synthase (PHB synthase) which comprise the three‐step PHB‐biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB‐biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl‐CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.
Error‐prone EF‐Tu reduces in vivo enzyme activity and cellular growth rate
Authors: Hughes D.
Published: October 2006

Mutations in Salmonella typhimurium encoding error‐prone EF‐Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF‐Tu‐ribo‐some interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error‐enhancing mutations are associated with a large reduction in the specific activity of a test protein, β‐galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error‐containing proteins.
A copy‐number mutant of plasmid pSC101
Authors: Xia G.‐X. , Manen D. , Goebel T. , Linder P. , Churchward G. , Caro L.
Published: October 2006

Copy‐number mutants of plasmid pSC101 were isolated by u.v. mutagenesis and selection for elevated expression of ampicillin resistance. Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA. The mutated plasmids were maintained at a level four to five times higher than that of the wild type. For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild‐type protein. We postulate that the copy‐number effect is due to a combination of these two effects, i.e. higher protein concentration and increased affinity of the protein for the repeated sequences.
Gas vesicle synthesis in the cyanobacterium Pseudanabaena sp.: occurrence of a single photoregulated gene
Authors: Damerval T. , Castets A.‐M. , Houmard J. , de Marsac N. Tandeau
Published: October 2006

Gas vesicles are subcellular inclusions found in a large number of aquatic prokaryotes. The gvpA gene, which frequently occurs as a multigene family, encodes the major gas vesicle structural protein. In several cyanobacteria, another gene, gvpC, encodes a different protein which might be a dispensable element for gas vesicle formation. We report here the molecular characterization of a gvpA gene in Pseudanabaena sp. PCC 6901. In this planktonic cyanobacterium, it is the only gvp gene which could be detected, and electrophoretic analysis of isolated gas vesicles revealed the presence of a single protein. A monocistronic mRNA species corresponds to the transcription of the gvpA gene and the abundance of the gvpA mRNA is inversely correlated with photosynthetic photon flux density, indicating that a light‐dependent transcriptional regulation is likely to be involved in the control of gas vacuolation in this strain.
Osmotic induction of the periplasmic trehalase in Escherichia coli K12: characterization of the treA gene promoter
Authors: Repoila F. , Gutierrez C.
Published: October 2006

The Escherichia coli treA gene encodes an osmotically inducible periplasmic trehalase. A strain carrying a treA–lacZ transcriptional fusion was constructed. The β‐galactosidase activity produced in this strain growing exponentially in a medium of high osmotic pressure was 10‐fold higher than that produced in a medium of low osmotic pressure, demonstrating that treA transcription is osmotically inducible. treA transcriptional induction depends neither on the presence of trehalase itself nor on the synthesis of cytoplasmic trehalose which occurs in response to osmotic stress in wild‐type E. coli strains. The treA promoter was identified by S1 nuclease protection. Deletion analysis demonstrated that sequences sufficient for the osmotic induction lie downstream from nucleotide –40 with respect to the transcription start. Transcription initiation at treAp required the presence of a functional σ70 subunit of RNA polymerase. treA expression was increased in the presence of a mutation in osmZ, which was previously identified as leading to a partially constitutive expression of the osmotically inducible proU operon.
The Escherichia coli cell cycle: one cycle or multiple independent processes that are co‐ordinated?
Authors: Nordstrom K. , Bernander R. , Dasgupta S.
Published: October 2006

In the life cycle of a bacterium there are several key processes: cellular growth, chromosome replication and decatenation, nucleoid partition, septum formation, and cell division. These processes have to be carefully controlled and co‐ordinated both with respect to each other and to the growth of the cell, and could be viewed as parts of a single cycle in which each step is dependent upon the previous one. Alternatively, they could be independently controlled and carefully tuned to each other without actually constituting a true cycle. In this review, using Escherichia coli as model system, we discuss these two ways of describing the bacterial life cycle. The evidence supporting independent control of the processes is presented, and some of the key questions in the elucidation of the regulation of the bacterial life cycle are discussed.
Cell division and peptidoglycan assembly in Eschenchia coli
Authors: Nanninga N.
Published: October 2006

Research on bacterial cell division has recently gained renewed impetus because of new information about peptidoglycan assembly and about specific cell‐division genes and their products. This paper concerns aspects of cell division that specifically concern the peptidoglycan. It is shown that upon division, peptidoglycan assembly switches from lateral wall location to the cell centre, that assembly takes place at the leading edge of the invaginating constriction, that the mode of glycan strand insertion changes from a single‐stranded mode to a multi‐stranded mode, and that the initiation of division (in contrast to its continuation) requires penicillin‐insensitive peptidoglycan synthesis (PIPS). A membrane component X (possibly FtsQ) is proposed to coordinate PIPS with the cell division‐initiating protein FtsZ. It is suggested that a largely proteinaceous macromolecular complex (divisome) at the leading edge of constriction encompasses three compartments (cytoplasm, membrane and periplasm). The composition of this complex is proposed to vary depending on whether division is being initiated or completed.
A suppressor of yeast spp81/ded1 mutations encodes a very similar putative ATP‐dependent RNA helicase
Authors: Jamieson D. J. , Beggs J. D.
Published: October 2006

The spp81/ded1 mutations were isolated as suppressors of a Saccharomyces cerevisiae pre‐mRNA splicing mutation, prp8‐1, The SPP81/DED1 gene encodes a isolated plasmids which were able to suppress the cold‐sensitive growth defect of SPP81 mutants. These plasmids encoded a gene (named DBP1) which mapped to chromosome XVI and not to the SPP81/DED1 locus on chromosome XV. The cloned gene suppressed the defect of SPP81/ded1 mutants when present on both high and low copy‐number plasmids but complemented SPP81/ded1 null mutants only when present on high copy‐number plasmids. In contrast to the SPP81/DED1 gene the DBP1 gene was not essential for cell viability. The nucleotide sequence of the DBP1 gene revealed that it also encoded a putative ATP‐dependent RNA helicase which showed considerable similarity at the amino acid level to the SPP81/DED1 protein.
Structure and function of peripiasmic chaperone‐like proteins involved in the biosynthesis of K88 and K99 fimbriae in enterotoxigenic Escherichia coli
Authors: Bakker D. , Vader C. E. M. , Roosendaal B. , Mooi F. R. , Oudega B. , de Graaf F. K.
Published: October 2006

The nucleotide sequence of faeE and fanE, two genes involved in the biosynthesis of K88 and K99 fimbriae, respectively, was determined and the amino acid sequence of the FaeE and FanE proteins was deduced. Immunobiotting of subcellular fractions with an anti‐serum raised against purified FaeE confirmed that FaeE is located in the periplasm. Indications were obtained that FaeE functions as a chaperone‐like protein, Its interaction with the fimbrial subunit (FaeG) in the periplasm stabilizes this polypeptide and prevents its degradation by the ceil‐envelope protease DegP. Furthermore, FaeE prevents the formation of FaeG multimers which cannot be incorporated into fimbriae. The reactions of the FaeE/FaeG dimers with a set of monoclonal antibodies directed against the various epitopes present on K88 fimbriae revealed that the fimbrial subunits associated with FaeE were present in a conformation resembling their native configuration. Indications about the domains in FaeG involved in the interaction with FaeE are discussed.
Temperature‐regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid
Authors: Tobe T. , Nagai S. , Okada N. , Adter B. , Yoshikawa M. , Sasakawa C.
Published: October 2006

The invasion phenotype of shigellae is subject to thermoregulation that is known to be expressed through activation of some invasion (inv) genes such as ipaB, ipaC, and ipaD encoded by the large virulence plasmid of Shigeila flexneri. The expression of ipa genes is regulated positively by virF through the activation of virB on the plasmid. To identify the mediator for the thermoregulation of the large plasmid, we have studied the effect of temperature on the transcription of virF and virB genes and ipa and the other two inv operons. The results showed that transcription of VirB was affected by temperature more strictly than that of virF. Analysis of the mRNA level of virB at different levels of virF transcription indicated that virB transcription depended upon both temperature and virF. On the other hand, transcriptions of ipa and the other two inv operons depended on the activation of virB transcription but not on temperature. By inducing wr8 transcription from a tac promoter fused with the virB region, invasion ability was restored to a WrF‐deletion mutant at 30°C as well as at 37°C. By using conditions in which the temperature‐dependent expression of the invasion phenotype was circumvented by the induction of virB transcription, intercellular spreading ability in a virF+, virB::Tn5 strain was shown to be expressed even at 30°C. These results suggest that the virB transcription stage is the main target for the thermoregulation.
Regulation of the chlA locus of Escherichia coli K12; involvement of molybdenum cofactor
Authors: Baker K. P. , Boxer D. H.
Published: October 2006

The chlA encodes functions required for the biosynthesis of the molybdopterin part of the molybdenum cofactor. Mutants, carrying gene fusions at the chlA locus, which place β‐galactosidase expression under the control of the chlA promoter, have been isolated employing λ plac Mui as the mutagen. The mutants exhibited β‐galactosidase expression which was greatly enhanced when grown anaerobically. Secondary mutations at the chlB, D, E or G loci did not affect the high level of expression. The fnr gene product was not required for the anaerobic expression. Bacteriophage λ transducing phages were isolated which carried the φ (chlA‐lac) mutations and were used to construct chlA/φ (chlA‐lac) merodiploids. The merodiploids exhibited a much lower level of expression but showed the same characteristics as strains carrying lac fusions to the single chromosomal chlA locus. Genetic evidence is presented which strongly suggests that the molybdenum cofactor is a repressor of chlA expression. The anerobic enhancement of chlA expression is mediated via a mechanism that is distinct from the molybdenum cofactor effect.
L‐pilin variants of Neisseria gonorrhoeae MS11
Authors: Manning P. A. , Kaufmann A. , Roll U. , Pohlner J. , Meyer T. F. , Haas R.
Published: October 2006

Phase‐ and antigenic variation of pilin expression in Neisseria gonorrhoeae is based on the genetic exchange between silent pilin genes (pilS)and the pilin expression locus (pilE). Similarly, the non‐piliated L‐variants of strain MS11, which show an increased resistance to certain antibiotics, are the result of recombination with the pilElocus. However, this recombination is atypical in that pilE(L) carries a tandem arrangement of a complete pilin gene and additional partial pilin genes under the control of the same pilE promoter. Since the two pilin gene copies are tandemly arranged and are often in the same translational frame, oversized pilin molecules are produced, which do not assemble into pili. The tandem gene copies introduced in a piiE(L)locus originate from silent loci where they are already joint. Upon reversion to the P+ phenotype the L‐variants lose one pilin gene copy from the pilE(L) in a process reminiscent of the deletion events that otherwise lead to the formation of the non‐revertible and non‐piliated Pn mutants of MS11 gonococci. Thus deletion of pilin genes from pHE can be regarded as a third mechanism of pilin variation in gonococci.
Insertional inactivation of the Staphylococcus aureusβ‐toxin by bacteriophage φ13 occurs by site‐and orientation‐specific integration of the φ 13 genome
Authors: Coleman D. , Knights J. , Russell R. , Shanley D. , Birkbeck T. H. , Dougan G. , Charles I.
Published: October 2006

Lysogenization of Staphylococcus aureus by the sero‐type F converting bacteriophage φ13 results in loss of β‐toxin expression. Sequence analysis of the S. aureusβ‐toxin gene (hlb), the attachment site (aftP)‐containing region of φ 13 DNA and the chromosome/bacteriophage DNA junctions of a φ13 lysogen, revealed that the molecular mechanism of loss of βtoxin expression was due to insertion of the φ13 genome into the 5′ end of hlb. The insertion site (attB) within hlb contained a 14 base pair core sequence in common with attP and both ends of the integrated linear prophage genome of a φ 13 lysogen. These findings indicate that integration of the φ13 genome into hlb is site‐and orientation‐specific.
Modular structure of Fix J: homology of the transcriptional activator domain with the ‐35 binding domain of sigma factors
Authors: Kahn D. , Ditta G.
Published: October 2006

The FixL/Fix J two‐component system is a global regulator of nitrogen‐fixation genes in Rhizobium meliloti. The transcriptional activator FixJ contains two modules: its N‐terminal module is homologous with other two‐component regulators; its C‐terminal module shows homology with various transcriptional activators, and with the C‐terminal region of σ factors, which is involved in the discrimination of the ‐35 region of bacterial promoters. We show that C‐terminal module of Fix J contains the entire transcription activation function, and that the N‐terminal module regulates this activity negatively. Oligonucleotide directed mutagenesis of the transcriptional activator module demonstrated the importance of a potential helix‐turn‐helix structure.
Fermentable sugars and intracellular acidification as specific activators of the RAS‐adenylate cyclase signalling pathway in yeast: the relationship to nutrient‐induced cell cycle control
Authors: Thevelein J. M.
Published: October 2006

The RAS proteins of the yeast Saccharomyces cerevisiae fulfil a similar control function on yeast adenylate cyclase as the mammalian Gs proteins on mammalian adenylate cyclase. The discovery that glucose and other fermentable sugars act as specific activators of the RAS‐adenylate cyclase pathway in yeast appeared to offer a mechanism for the way in which at least one nutrient would control progression over the start point in the G1 phase of the yeast cell cycle by means of this pathway. Recently, however, evidence has been obtained to show that the glucose‐activation pathway of adenylate cyclase is a glucose‐repressible pathway and therefore not operative during growth on glucose. In addition, mutant strains were obtained which lack the glucose‐activation pathway and show normal exponential growth on glucose. This appears to confine the physiological role of this pathway to control of the transition from the derepressed state (growth on respirative carbon sources) to the repressed state (growth on fermentative carbon sources) by means of an already well‐documented cAMP‐triggered protein phosphorylation cascade. Intracellular acidification also stimulates the RAS‐adenylate cyclase pathway, which might constitute a rescue mechanism for cells suffering from stress conditions. The presence of a nitrogen source does not stimulate the RAS‐adenylate cyclase pathway. Although other nutrient signals for the pathway might still be discovered, it appears more and more likely that the well‐known requirement of cAMP for progression over the start point of the yeast cell cycle is limited to providing a basal cAMP level rather than acting as a second messenger for an extracellular signal. A model In which a nitrogen‐source‐induced pathway leads to activation of cAMP‐dependent protein kinase synergistically with cAMP, or alternatively causes activation only of the free catalytic subunits, would provide an elegant explanation for the apparent contradiction that nitrogen‐source induced progression over the start point of the yeast cell cycle does not seem to be mediated by cAMP but on the other hand seems to depend on cAMP and cAMP‐dependent protein kinase.
Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron‐transport system: characterization of a gene cluster involved in uptake and biosynthetic pathways
Authors: Franza T. , Enard C. , van Gijsegem F. , Expert D.
Published: October 2006

Twenty of the twenty‐two MudII1734 insertions impairing the chrysobactin iron‐assimilation system of Erwinia chrysanthemi 3937 were localized to a 50kbp genomic insert contained in the R‐prime plasmid, R′4 (Enard et al., 1988). Using the conjugative plasmid pULB1 10 (RP4::mini‐Mu) and the generalized transducing phage ΦEC2, we located this iron‐transport region and the two unlinked mutations on the chromosome linkage map. Chrysobactin is a catechol‐type siderophore and, as we have previously observed with the entA locus of Escherichia coli, the E. chrysanthemi‐derived R′4 was found to complement E. coli entB and entE mutations. A 2.9 kb Eco Ri and a 4.8kb BamHI fragment in the R′4 sharing homology with the E. coli entCEBAP15 operon DNA were subcloned. These fragments were used as DNA/DNA hybridization probes to screen a wild‐type gene library, yielding a recombinant cosmid (pEC7) able to complement mutations disrupting the 2,3‐drhydroxy‐benzoic acid biosynthetic pathway in both Erwinia and Escherichia spp. as well as the E. coli entE mutation. Physical mapping of the genomic MudII1734 insertions corresponding to these mutations led to the identification of a cluster of genes confined to a DNA sequence of about 10 kb required for both biosynthetic and receptor functions.
Characterization of the opa (class 5) gene family of Neisseria meningitidis
Authors: Aho E. L. , Dempsey J. A. , Hobbs M. M. , Klapper D. G. , Cannon J. G.
Published: October 2006

Class 5 outer membrane proteins of Neisseria meningitidis show both phase‐ and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used λgt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.
Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants
Authors: Blomfjeld I. C. , McClain M. S. , Eisenstein B. I.
Published: October 2006

We have used Southern hybridization analysis to characterize the extent of fim homology in recognized type 1 fimbriae mutants of Escherichia coli K12, including strains HB101, P678‐54, and VL584. We have found extensive homology in strain HB101, and confirm that P678‐54 lacks the majority of fim DNA. Strain VL584 contains a deletion of the entire fim region. We have used a new allelic exchange procedure to generate novel fim deletion derivatives of strains MG1655, MM294, and YMC9. To increase the utility of the new deletion strains we also isolated recA derivatives of each mutant. These strains facilitate the isolation, characterization, and manipulation of cloned fimbriae genes from diverse sources.
Enhancement of the invasive ability of Neisseria gonorrhoeae by contact with HeclB, an adeno‐carcinoma endometrial cell line
Authors: Chen J. C.‐R. , Bavoil P. , Clark V. L.
Published: October 2006

Since Neisseria gonorrhoeae is an obligate pathogen, there is no animal model for identification of virulence factors for this bacterium. An alternative model for assessment of gonococcal virulence is invasion of the adenocarcinoma endometrial cell line, HeclB. Pre‐incubation of gonococci with glutaraldehyde‐fixed HeclB cells eliminated the six‐ to eight‐hour lag in entry of bacteria into a fresh HeIIB monolayer seen with unpreincubated gonococci or gonococci pre‐incubated in tissue‐culture medium alone. Gonococci tightly bound to fixed HecIB cells were more Invasive than cells free in the tissue‐culture medium, suggesting that actual contact with HecIB cells was required for the enhancement of invasive ability. Chloramphenicol addition during the preincubation prevented the enhanced invasion. Preincubated gonococci were not more adherent to HecIB cells, suggesting that a stage in invasion after binding of gonococci to HecIB cells was enhanced. The enhanced invasion occurred only when gonococci were preincubated with HecIB ceils and not with HEp‐2, HeLa, Chang or CHO cells. This eukaryotic cell specificity for induction of enhanced invasion may indicate a role for invasion in gonococcal infection of the endometrium.
Regulation of retrotransposition in Saccharomyces cerevisiae
Authors: Curcio M. J. , Garfinkel D. J.
Published: October 2006

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at at a rate of 10−5 to 10−7 per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.
A Bacillus subtilis dipeptide transport system expressed early during sporulation
Authors: Mathiopoulos C. , Mueller J. P. , Slack F. J. , Murphy C. G. , Patankar S. , Bukusoglu G. , Sonenshein A. L.
Published: October 2006

Two previously identified Bacillus subtilis DNA segments, dciA and dciB, whose transcripts accumulate very rapidly after induction of sporulation, were found in the same 6.2 kb transcription unit, now known as the dciA operon. Analysis of the sequence of the dciA operon showed that its putative products are homologous to bacterial peptide transport systems. The product of the fifth gene, DciAE, is similar to peptide‐binding proteins from Escherichia coli and Salmonella typhimurium (DppA and OppA) and B. subtilis (OppA). A null mutation in dciAE abolished the ability of a proline auxotroph to grow in a medium containing the dipeptide Pro–Gly as sole proline source, suggesting that the dciA operon encodes a dipeptide transport system.
Transcriptional regulation of a Bacillus subtilis dipeptide transport operon
Authors: Slack F. J. , Mueller J. R , Strauch M. A. , Mathiopoulos C. , Sonenshein A. L.
Published: October 2006

The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post‐exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB.
Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK‐like gene, a positive regulator of nifA
Authors: Kaminski P. A. , Mandon K. , Arigoni F. , Desnoues N. , Elmerich C.
Published: October 2006

The nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established. An open reading frame of 744 bp was identified as a fixK homologue. A kanamycin cartridge was inserted into the cloned fixK‐1ike gene and recombined Into the host genome. The resulting mutant was Nif− Fix− suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free‐living state. Using a pfixK–lacZ fusion, the FixLJ products were shown to control the expression of fixK. Using a pnifA–lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free‐living state. In addition, a double ntrC–fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity. A model of regulation, based on these data, is presented and might explain the unusual ability of A. caulinodans to fix nitrogen both under symbiotic conditions and in the free‐living state.
Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin‐resistant clinical isolates of Streptococcus pneumoniae
Authors: Laible G. , Spratt B. G. , Hakenbeck R.
Published: October 2006

Penicillin resistance in pneumococci is due to the appearance of high molecular‐weight penicillin‐binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX2x genes (pbpX) of one penicillin‐susceptible and five penicillin‐resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low‐affinity form of PBP 2x. The 2kb region of the two penicillin‐susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (<3% diverged), alternating with regions that were very different (18–23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson etal., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to β‐lactam antibiotics. Indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.
Bacillus subtilis chytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa3‐type oxidases
Authors: van der Oost J. , von Wachenfeld C. , Hederstedt L. , Saraste M.
Published: October 2006

The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene‐replacement using a phleomycin‐resistance marker. The mutant was unable to oxidize N,N,N′,N′‐tetramethyl‐p‐phenylene‐diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild‐type membranes confirmed the presence of two haem A‐containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem‐labelling was used to identify sub‐unit ii, which contains a haem C, and cytochrome c‐550 among the membrane‐bound c‐type cytochromes of B. subtilis.
Molecular chaperones and protein translocation across the Escherichia coli inner membrane
Authors: Kumamoto C.A.
Published: October 2006

Proteins that are able to translocate across biological membranes assume of loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as‘per‐folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre‐folded conformation and have been termed‘molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.
Identification of a central regulator of stationary‐phase gene expression in Escherichia coli
Authors: Lange R. , Hengge‐Aronis R.
Published: October 2006

During carbon‐starvation‐induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30–50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C‐starvation‐regulated genes we isolated and characterized chromosomal C‐starvation‐ induced csi::lacZ fusions using the λplac Mu system. One operon fusion (csi 2::lacZ) has been studied in detail. csi 2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two‐dimensional gel electrophoresis, cells carrying csi 2::lacZ did not synthesize at least 16 proteins present In an isogenic csi 2+ strain. Cells containing csi 2::lacZ or csi 2::Tn10 did not produce glycogen, did not develop thermo‐tolerance and H2O2 resistance, and did not induce a stationary‐phase‐specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild‐type cells during prolonged starvation. We conclude that csi 2::lacZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild‐type gene corresponding to csi 2 demonstrated that the csi 2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi 2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose ‘rpoS’ (‘σs’) as appropriate designations.
Repression and derepression of conjugation of plasmid R1 by wild‐type and mutated finP antisense RNA
Authors: Koraimann G. , Koraimann C. , Koronakis V. , Schlager S. , Högenauer G.
Published: October 2006

The finP gene of plasmid R1 is located between the genestraM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5′ non‐translated sequences, the ribosome‐binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1‐19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R l‐19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1‐19 intrans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.
Virulence‐associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis
Authors: Okada N. , Sasakawa C. , Tobe T. , Yamada M. , Nagai S. , Talukder K. A. , Komatsu K. , Kanegasaki S. , Yoshikawa M.
Published: October 2006

Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the IpaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 Notl restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence‐associated chromosomal loci.
Leader peptidase
Authors: Dalbey R. E.
Published: October 2006

The Escherichia coli leader peptidase has been vital for unravelling problems in membrane assembly and protein export. The role of this essential peptidase is to remove amino‐terminal leader peptides from exported proteins after they have crossed the plasma membrane. Strikingly, almost ail periplasmic proteins, many outer membrane proteins, and a few inner membrane proteins are made with cleavable leader peptides that are removed by this peptidase. This enzyme of 323 amino acid residues spans the membrane twice, with its large carboxyl‐terminal domain protruding into the periplasm. Recent discoveries show that its membrane orientation is controlled by positively charged residues that border (on the cytosolic side) the transmembrane segments. Cleavable pre‐proteins must have small residues at –1 and a small or aliphatic residue at –3 (with respect to the cleavage site). Leader peptidase does not require a histidine or cysteine amino acid for catalysis. Interestingly, serine 90 and aspartic acid 153 are essential for catalysis and are also conserved in a mitochondrial leader peptidase, which is 30.7% homologous with the bacterial enzyme over a 101‐residue stretch.
Alternative sigma factors and the regulation of flagellar gene expression
Authors: Helmann J. D.
Published: October 2006

Synthesis of bacterial flagella and the accompanying array of chemotaxis receptors and transducers represents a major commitment of energy and resources for a growing bacterial cell and is subject to numerous levels of regulation. Genes for flagellar and chemotaxis proteins are expressed in a complex transcriptional cascade. This regulatory hierarchy acts to ensure that the highly expressed filament structural protein, flagellin, is synthesized only after a prerequisite set of structural proteins has been expressed and properly assembled. Recent evidence suggests that many bacteria utilize an alternative sigma (σ) subunit, similar in specificity to the Bacillus subtilisσ28 protein, to direct transcription of flagellin, chemotaxis and motility genes. In Caulobacter crescentus and Campylobacter spp., both a σ54‐like factor and a σ28‐like factor participate in the transcription of flagellar and chemotaxis genes. Conversely, a σ28‐like factor controls non‐motility functions in at least one non‐flagellated organism.
Evidence for a TonB‐dependent energy transduction complex in Escherichia coli
Authors: Skare J. T. , Postle K.
Published: October 2006

Escherichia coli TonB protein is required for the active transport of vitamin B12 and Fe(III)‐siderophore complexes across the outer membrane, infection by bacteriophages T1 and φ80, and sensitivity to B‐group colicins. TonB appears to function as an energy transducer in these processes, coupling cytoplasmic membrane electrochemical potential to receptors in the outer membrane. Previous reports have demonstrated that chromosomally encoded TonB is functionally unstable in the absence of protein synthesis (half‐life ∼15–30 minutes) and have shown that plasmid‐encoded, overexpressed TonB is chemically unstable (half‐life ∼5 minutes). In contrast, this study has shown that chromosomally encoded TonB was chemically stable for greater than 90 minutes while maintaining its functional instability. These data suggest that proteolytic degradation of TonB protein is not the basis of its functional instability. Auxiliary proteins such as ExbB also play a role in TonB‐dependent energy transduction. In this study, we have shown that the chemical half‐life of chromosomally encoded TonB in an exbB::Tn10 mutant was reduced at least 18‐fold, suggesting that TonB is a part of a cytoplasmic membrane complex that includes, at the minimum, ExbB. These results also suggest that the chemical instability of plasmid‐encoded TonB resulted when the TonB ExbB ratio was too high and are consistent with previous observations that plasmid‐encoded ExbB can stabilize plasmid‐encoded TonB. The exbB mutation also resulted in a significant decrease in TonB function as measured by the ability of cells to adsorb bacterio‐phage Φ80. In a previous study, strains carrying a tolQ nonsense mutation in combination with an exbB mutation were observed to mimic a tonB phenotype, suggesting that either ExbB or TolQ can alternatively activate TonB. In contrast, we have shown that neither the chemical half‐life nor the function of TonB are affected by the tolQ mutation, and thus, unlike ExbB, TolQ plays a minimal role in TonB‐dependent processes.
Characterization of an oxytetracycline‐resistance gene, otrA, of Streptomyces rimosus
Authors: Doyle D. , McDowall K. J. , Butler M. J. , Hunter I. S.
Published: October 2006

The sequence of a 2657 bp DNA fragment containing the coding and regulatory regions of the oxytetracycline (OTC)‐resistance gene, otrA, from the OTC producer Streptomyces rimosus was determined. The predicted amino acid sequence of OtrA had extensive identity with tetracycline‐resistance genes from other bacteria which mediate resistance via non‐covalent ribosomal modification. The N‐terminal domain had extremely high identity with the GTP‐binding sites of elongation factors, such as EF‐G and EF‐Tu, suggesting that binding and hydrolysis of GTP is important to the function of the protein. Significant identity with EF‐G was present throughout the polypeptide. Transcriptional activity upstream of the otrA coding region was investigated. An Escherichia coli‐ type promoter, of otrA p1, was identified. Transcriptional read through of otrA from the upstream gene (otcZ) was also detected in S. rimosus cultures. A divergent promoter activity was identified with sub‐clones of the OtrA fragment in promoter probe vectors analysed in Streptomyces lividans. However, this activity was not identified in a subclone containing more than haif of the otrA coding sequence in S. lividans or at all in S. rimosus, indicating that OtrA negatively regulates the expression of the divergent transcript. The data are consistent with regulation of antibiotic production by OtrA to prevent ‘suicide’.
RNaselll activation of bacteriophage λ N synthesis
Authors: Kameyama L. , Fernandez L. , Court D. L. , Guarneros G.
Published: October 2006

The bacteriophage λ gene product is one of the first genes expressed during phage development. N protein allows the expression of other phage genes by altering the transcription elongation process so as to prevent transcription termination. We have found that N levels may be modulated soon after induction or infection. Using N‐lacZ fusions, we determined that cells containing RNaselll have at least a fourfold greater expression than cells defective for RNaselll. This effect is exerted at the post‐transcriptional level. RNaselll processes an RNA stem structure in the N‐ leader RNA. Removal of the stem structure by deletion increases λ expression and prevents further stimulation by RNaselll. The base of this stable stem is adjacent to the λ ribosome binding site. We present a model for control of N synthesis in which this stable stem inhibits ribosome access to the N mRNA.
A host factor absent from Lactococcus lactis subspecies lactis MG1363 is required for conjugative transposition
Authors: Bringel F. , Van Alstine G. L. , Scott J. R.
Published: October 2006

In matings between Lactococcus lactis strains, the conjugative transposons Tn916 and Tn919are found in the chromosome of the transconjugants in the same place as in the chromosome of the donor, indicating that no transposition has occurred. In agreement with this, the frequency of L. lactis transconjugants from intraspecies matings is the same whether the donor contains the wild‐type form of the transposon or the mutant Tn916‐int1, which has an insertion in the transposon's integrase gene. However, in intergeneric crosses with Bacillus subtilis or Enterococcus faecalis donors, Tn916 and Tn919 transpose to different locations on the chromosome of the L. lactis transconjugants. Moreover, Tn916 and Tn919 could not be transferred by conjugation from L. lactis and B. subtilis, E. faecalis or Streptococcus pyogenes. This suggests that excision of these elements does not occur in L. lactis. When cloned into E. coli with adjacent chromosomal DNA from L. lactis, the conjugative transposons were able to excise, transpose and promote conjugation. Therefore, the inability of these elements to excise in L. lactis is not caused by a permanent structural alteration in the transposon. We conclude that L. lactis lacks a factor required for excision of conjugative transposons.
Molecular cloning and transcriptional analysis of the cpeBA operon of the cyanobacterium Pseudanabaena species PCC 7409
Authors: Dubbs J. M. , Bryant D. A.
Published: October 2006

The cpeBA operon of the Group III chromatically adapting cyanobacterium Pseudanabaena species PCC 7409 was cloned, sequenced and characterized. The cpeBA genes are transcribed in green‐light‐grown cells as an abundant 1400‐nucleotide mRNA which initiates 69 nucleotides upstream from the cpeB translation start. Extensive sequence identity, extending 70 nucleotides 5′ to the transcription start, occurs among cpeBA promoters of Group II and III chromatic adapters. Cell extracts of green‐light‐grown Calothrix species PCC 7601 contain an activity which specifically binds a restriction fragment containing the Pseudanabanea species PCC 7409 cpeBA promoter. Green‐light‐dependent cpeBA transcription in Group II and III chromatically adapting cyanobacteria is suggested to be similarly controlled by a transcriptional activator.
The role of RNA polymerase in transcriptional fidelity
Authors: Libby R. T. , Gallant J. A.
Published: October 2006

The overall transcription of DNA has previously been demonstrated to proceed at extremely high levels of accuracy. We review the evidence that the process of transcription is subject to proof‐reading in the Hopfield sense. In addition, we speculate that the proof‐reading activity associated with transcription is subject to cyclical phase transitions. That is, during periods of low processivity associated with initiation, RNA synthesis is relatively imprecise. The transition to the elongation phase of RNA synthesis, characterized by a shift to high processivity, is accompanied by enhanced proof‐reading. A model for the damping of transcriptional errors, based on a PPi‐mediated processive pyrophosphorolysis reaction, is discussed in terms of pausing during transcription.
Molecular analysis of a complex locus from Haemophilus influenzae invoked in phase‐variable lipopolysaccharide biosynthesis
Authors: Maskell D. J. , Szabo M. J. , Butler P. D. , Williams A. E. , Moxon E. R.
Published: October 2006

A chromosomal locus, lic3, one of several involved in lipopolysaccharide (LPS) biosynthesis by Haemophilus influenzae, was cloned and its DNA sequence determined. Iic3 comprises four closely apposed open reading frames (ORFs). ORF1 includes tandem repeats of the tetramer CAAT and two start codons out of frame with each other are found upstream of the repeats. ORF1 encodes a protein with no known homologues. 0RF2 encodes the UDP‐galactose‐4‐epimerase (galE) gene. ORF3 encodes a hydrophobic protein with no known homologues. 0RF4 encodes the adenylate kinase (adk) gene. A deletion/insertion mutation lacking the 3′ end of ORF1, all of galE, and the 5′ end of ORF3 was constructed in the parent Hib strain (RM7004). These mutants had a galE phenotype, as evidenced by galactose sensitivity, altered LPS when grown in the absence of exogenous galactose, and reduced virulence in infant rats.