bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of directed motility in Myxococcus xanthus
Authors: Ward Mandy J. , Zusman David R.
Published: November 2003

Myxococcus xanthus is a Gram‐negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the ‘frizzy’ signal‐transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY‐like response‐regulator domains: one is present on FrzE, which also contains a CheA‐like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self‐generated autoattractants. The Frz signal‐transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting‐body development and vegetative swarming.
Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity
Authors: Guina Tina , Oliver Donald B.
Published: November 2003

We cloned the gene encoding a membrane‐interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte‐species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four‐gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted α‐helical 7.4 kDa protein with a hydrophobic central region and a positively charged C‐terminus, which is structurally homologous to a large group of pore‐forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane‐associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X‐114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.
Identification of Pseudomonas aeruginosa genes required for epithelial cell injury
Authors: Jung Kang Pil , Hauser Alan R. , Apodaca Gerard , Fleiszig Suzanne M. J. , Wiener‐Kronish Jeanine , Mostov Keith , Engel Joanne N.
Published: November 2003

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa‐induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon‐tagged non‐cytotoxic mutants of a cytotoxic and lung‐virulent strain of P. aeruginosa (PA103). The transposon‐insertion site was determined by using an inverse polymerase chain reaction followed by DNA‐sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt−). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein‐secretion apparatus appears to be involved in this process.
Osmostress response in Bacillus subtilis: characterization of a proline uptake system (OpuE) regulated by high osmolarity and the alternative transcription factor sigma B
Authors: Von Blohn Carsten , Kempf Bettina , Kappes Rainer M. , Bremer Erhard
Published: November 2003

Exogenously provided proline has been shown to serve as an osmoprotectant in Bacillus subtilis. Uptake of proline is under osmotic control and functions independently of the known transport systems for the osmoprotectant glycine betaine. We cloned the structural gene (opuE ) for this proline transport system and constructed a chromosomal opuE mutant by marker replacement. The resulting B. subtilis strain was entirely deficient in osmoregulated proline transport activity and was no longer protected by exogenously provided proline, attesting to the central importance of OpuE for proline uptake in high‐osmolarity environments. The transport characteristics and growth properties of the opuE mutant revealed the presence of a second proline transport activity in B. subtilis. DNA sequence analysis of the opuE region showed that the OpuE transporter (492 residues) consists of a single integral membrane protein. Database searches indicated that OpuE is a member of the sodium/solute symporter family, comprising proteins from both prokaryotes and eukaryotes that obligatorily couple substrate uptake to Na+ symport. The highest similarity was detected to the PutP proline permeases, which are used in Escherichia coli, Salmonella typhimurium and Staphylococcus aureus for the acquisition of proline as a carbon and nitrogen source, but not for osmoprotective purposes. An elevation of the osmolarity of the growth medium by either ionic or non‐ionic osmolytes resulted in a strong increase in the OpuE‐mediated proline uptake. This osmoregulated proline transport activity was entirely dependent on de novo protein synthesis, suggesting a transcriptional control mechanism. Primer extension analysis revealed the presence of two osmoregulated and tightly spaced opuE promoters. The activity of one of these promoters was dependent on sigma A and the second promoter was controlled by the general stress transcription factor sigma B.
Unsuspected prophage‐like elements in Salmonella typhimurium
Authors: Figueroa‐Bossi Nara , Coissac Eric , Netter Pierre , Bossi Lionello
Published: November 2003

We present evidence for the existence of two large (≈50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements — designated Gifsy‐1 and Gifsy‐2 — cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy‐1 yield the classical ‘Sbc’ phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE‐like gene. At the same time, these ‘sbcE ’ mutations activate a Xis‐type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy‐1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy‐2 even though the Gifsy‐1‐associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy‐1 drops dramatically in Gifsy‐2‐cured cells. Thus, both elements must co‐operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy‐1 and Gifsy‐2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).
Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion
Authors: Lu Hong‐Mei , Motley S.Timothy , Lory Stephen
Published: November 2003

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin‐like Xcp proteins was investigated using bifunctional cross‐linking reagents. Cross‐linking analysis of whole cells of wild‐type P. aeruginosa, followed by immunoblot analysis, revealed a 34‐kDa XcpT‐containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PilC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N‐terminus of PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be cross‐linked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine‐tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co‐purified by nickel‐affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.
PilC of pathogenic Neisseria is associated with the bacterial cell surface
Authors: Rahman Motiur , Källström Helena , Normark Staffan , Jonsson Ann‐Beth
Published: November 2003

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high‐molecular‐weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1− mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus‐mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non‐piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.
The use of flash photolysis for a high‐resolution temporal and spatial analysis of bacterial chemotactic behaviour: CheZ is not always necessary for chemotaxis
Authors: Dowd John P. , Matsumura Philip
Published: November 2003

The purpose of this work was to develop a high‐resolution analysis of behaviour as an assay of the physiological consequences of mutations in the che genes and also to examine the role of CheZ in chemotaxis. Recent advances in flash photolysis have made it possible to expose cells to an unstable chemical gradient created by a square‐wave increase in attractant concentration. The response of individual cells can be tracked in the order of milliseconds using real‐time motion analysis. The tumble frequency of wild‐type Escherichia coli exposed to photoreleased aspartate falls very quickly to smooth‐swimming levels, and the swimming speed of these cells rises. As a consequence of these behavioural changes, there is an increase in the number of bacteria present in the centre of the flashed area, that is the bacteria’s response to the transient gradient generated by flash photolysis was to swim into the centre of the flash area. This allowed the rapid quantitative measurement of chemotaxis. Deletion of various che genes resulted in predictable changes in chemotactic behaviour. cheZ null mutants are non‐chemotactic when measured by classical techniques but demonstrate a definite chemotactic response to photoreleased attractant.
Inactivation of the 20S proteasome in Mycobacterium smegmatis
Authors: Knipfer Nancianne , Shrader Thomas E.
Published: November 2003

The 20S proteasome is an essential component of the cytosolic protein turnover apparatus of eukaryotic cells. In higher eukaryotes, the 20S proteasome is responsible for most cytosolic protein turnover and also generates peptides for subsequent presentation by the MHC class I pathway. Structurally, the eukaryotic 20S proteasome is extremely complex, being composed of 14 different subunits. Proteasomes with simplified subunit composition have been identified in certain eubacteria and archaebacteria but, in each case, the proteasome‐containing organism is recalcitrant to further molecular genetic analyses. As a result, no in vivo characterization of a simplified eubacterial or archaebacterial proteasome has been reported. We have shown that the genetically tractable eubacterium Mycobacterium smegmatis contains a 20S proteasome, allowing the first in vivo characterization of a simplified 20S proteasome. We use a positive/negative selection scheme to inactivate the genes encoding 20S proteasome subunits and demonstrate that, in contrast to eukaryotic cells, M. smegmatis cells lacking intact proteasome genes are viable and phenotypically indistinguishable from congenic strains containing proteasomes. Implications for the evolution of the protein turnover apparatus are discussed.
In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing σA, the major sigma factor in growing cells
Authors: Biran Dvora , Kroos Lee
Published: November 2003

Myxococcus xanthus is a Gram‐negative bacterium that undergoes multicellular development upon starvation. We have developed a simple and rapid procedure for partial purification of RNA polymerase from growing M. xanthus cells, using heparin–agarose and DNA–cellulose chromatographies. In addition to core subunits, the enzyme contains one fairly abundant polypeptide of approximately 105 kDa. We have shown by Western blot analysis and protein sequencing that the 105‐kDa polypeptide is σA, the product of the M. xanthus sigA gene. Partially purified σA RNA polymerase, or holoenzyme reconstituted from σA and core RNA polymerase, transcribed in vitro the vegA and aphII genes that are known to be expressed in growing M. xanthus cells. Reconstituted σA RNA polymerase produced vegA mRNA in vitro with the same 5′ end as vegA mRNA produced in vivo, demonstrating that initiation of transcription was accurate in vitro. These results provide biochemical evidence that σA is the major vegetative sigma factor of M. xanthus. To our knowledge, this is the first report of in vitro transcription of M. xanthus chromosomal genes, providing a foundation for further biochemical analysis of transcriptional regulatory mechanisms in a microbe that relies extensively on cell–cell interactions.
Altered ParA partition proteins of plasmid P1 act via the partition site to block plasmid propagation
Authors: Youngren Brenda , Austin Stuart
Published: November 2003

The partition system of the P1 plasmid, P1parconsists of the ParA and ParB proteins and a cis‐acting site, parS. It is responsible for the orderly segregation of plasmid copies to daughter cells. Plasmids with null mutations in parA or parB replicate normally, but missegregate. ParB binds specifically to the parS site, but the role of ParA and its ATPase activity in partition is unclear. We describe a novel class of parA mutants that cannot be established or maintained as plasmids unless complemented by the wild‐type gene. One, parAM314I, is conditional: it can be maintained in cells in minimal medium but cannot be established in cells growing in L broth. The lack of plasmid propagation in L broth‐grown cells was shown to be caused by a ParB‐dependent activity of the mutant ParA protein that blocks plasmid propagation by an interaction at the parS site. Thus, ParA acts to modify the ParB–parS complex, probably by binding to it. Partition is thought to involve selection of pairs of plasmids before segregation, either by physical pairing of copies or by binding of copies to paired host sites. We suggest that ParA is involved in this reaction and that the mutant ParA protein forms paired complexes that cannot unpair.
Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria
Authors: Källström Helena , Liszewski M. Kathryn , Atkinson John P. , Jonsson Ann‐Beth
Published: November 2003

Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cell‐surface receptors. We found that purified pili bound to a 55‐ to 60‐kDa doublet band on SDS–PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria. We proved this hypothesis by demonstrating that piliated, but not non‐piliated, gonococci bound to CHO cells transfected with human MCP‐cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria. Finally, recombinant MCP protein produced in E. coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell‐surface receptor for piliated pathogenic Neisseria.
Regulation of inositol monophosphatase in Saccharomyces cerevisiae
Authors: Murray Marlene , Greenberg Miriam L.
Published: November 2003

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second‐messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium’s pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro. Growth in the presence of inositol resulted in increased expression of the enzyme in vivo, although inositol had no effect on enzyme activity in vitro. The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.
ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury
Authors: Finck‐Barbançon Viviane , Goranson Joanne , Zhu Lei , Sawa Teiji , Wiener‐Kronish Jeanine P. , Fleiszig Suzanne M. J. , Wu Christine , Mende‐Mueller Liane , Frank Dara W.
Published: November 2003

The production of exoenzyme S is correlated with the ability of Pseudomonas aeruginosa to disseminate from epithelial colonization sites and cause a fatal sepsis in burn injury and acute lung infection models. Exoenzyme S is purified from culture supernatants as a non‐covalent aggregate of two polypeptides, ExoS and ExoT. ExoS and ExoT are encoded by separate but highly similar genes, exoS and exoT. Clinical isolates that injure lung epithelium in vivo and that are cytotoxic in vitro possess exoT but lack exoS, suggesting that ExoS is not the cytotoxin responsible for the pathology and cell death measured in these assays. We constructed a specific mutation in exoT and showed that this strain, PA103 exoT::Tc, was cytotoxic in vitro and caused epithelial injury in vivo, indicating that another cytotoxin was responsible for the observed pathology. To identify the protein associated with acute cytotoxicity, we compared extracellular protein profiles of PA103, its isogenic non‐cytotoxic derivative PA103 exsA::Ω and several cytotoxic and non‐cytotoxic P. aeruginosa clinical isolates. This analysis indicated that, in addition to expression of ExoT, expression of a 70‐kDa protein correlated with the cytotoxic phenotype. Specific antibodies to the 70‐kDa protein bound to extracellular proteins from cytotoxic isolates but failed to bind to similar antigen preparations from non‐cytotoxic strains or PA103 exsA::Ω. To clone the gene encoding this potential cytotoxin we used Tn5Tc mutagenesis and immunoblot screening to isolate an insertional mutant, PA103exoU:: Tn5Tc, which no longer expressed the 70‐kDa extracellular protein but maintained expression of ExoT. PA103 exoU::Tn5Tc was non‐cytotoxic and failed to injure the epithelium in an acute lung infection model. Complementation of PA103exoU::Tn5Tc with exoU restored cytotoxicity and epithelial injury. ExoU, ExoS and ExoT share similar promoter structures and an identical binding site for the transcriptional activator, ExsA, data consistent with their co‐ordinate regulation. In addition, all three proteins are nearly identical in the first six amino acids, suggesting a common amino terminal motif that may be involved in the recognition of the type III secretory apparatus of P. aeruginosa.
Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product
Authors: Katayama Tsutomu , Akimitsu Nobuyoshi , Mizushima Tohru , Miki Takeyoshi , Sekimizu Kazuhisa
Published: November 2003

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli, is regulated by adenine nucleotide binding; the ATP‐bound form, not the ADP‐bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30°C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn5 insertion mutagenesis. Three of the suppressors carried Tn5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over‐replication of the chromosome was inhibited in the dnaAcos aroK::Tn5 double mutant, and initiation of chromosome replication in the dnaA+aroK::Tn5 mutant was partially inhibited. The aroK (or B )::Tn5 cells contained DnaA molecules at a level similar to that in the parental aroBK + strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo. SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.
A motile but non‐swarming mutant of Proteus mirabilis lacks FlgN, a facilitator of flagella filament assembly
Authors: Gygi Daniel , Fraser Gillian , Dufour Alain , Hughes Colin
Published: November 2003

A TnphoA mutant of Proteus mirabilis was isolated, which had lost the ability to swarm, yet was still motile. The transposon had inserted into flgN, a flagella gene encoding a 147‐amino‐acid protein of undefined function. Proteus flgN is arranged in an operon with the class III anti‐σ28 gene, flgM, flanked by the class II genes, flgA, flgBCD and flhBA, and a novel putative virulence‐related gene. The flgN mutation caused a substantial reduction in cell surface‐associated flagellin, particularly during differentiation to the normally hyperflagellated swarm cell. This was not due to an effect on flagella gene expression or a typical defect in the flagella export apparatus as there was no class III gene downregulation by FlgM feedback, or intracellular flagellin accumulation. Loss of FlgN nevertheless caused a severe reduction in the incorporation of pulse‐labelled flagellin into the membrane/flagellum fraction of differentiating cells. Substantial amounts of both non‐oligomeric flagellin and flagellin degradation products appeared in the extracellular medium, although the few mature filaments made by the mutant were no more sensitive to proteolysis than those of the wild type. FlgN appeared soluble and active in the cytosol. The data suggest that the function of FlgN is to facilitate the initiation of flagella filament assembly, a role that may be especially critical in attaining the much higher concentration of surface flagellin required for swarming. Proteus FlgN has leucine zipper‐like motifs arranged on potential amphipathic helices, a feature conserved in cytosolic chaperones for the exported substrates of flagella‐related type III virulence systems. While gel filtration of FlgN from the soluble cell fraction did not establish an interaction with flagellin, it indicated that FlgN may associate with an unknown component and/or form an oligomer.
Messenger RNA release from ribosomes during 5′‐translational blockage by consecutive low‐usage arginine but not leucine codons in Escherichia coli
Authors: Gao Wenwu , Tyagi Sanjay , Kramer Fred Russell , Goldman Emanuel
Published: November 2003

In ‘5′‐translational blockage’, significantly reduced yields of proteins are synthesized in Escherichia coli when consecutive low‐usage codons are inserted near translation starts of messages (with reduced or no effect when these same codons are inserted downstream). We tested the hypothesis that ribosomes encountering these low‐usage codons near the translation start prematurely release the mRNA. RNA from polysome gradients was fractionated into pools of polysomes and monosomes and a ribosome‐free pool. New hybridization probes, called ‘molecular beacons’, and standard slot blots were used to detect test messages containing either consecutive low‐usage AGG (arginine) or synonymous high‐usage CGU insertions near the 5′ end. The results show an approximately twofold increase in the ratio of free to bound mRNA when the low‐usage codons were present in the message compared with when high‐usage codons were present. In contrast, there was no difference in the ratio of free to bound mRNA when consecutive low‐usage CUA or high‐usage CUG (leucine) codons were inserted or when the arginine codons were inserted near the 3′ end. These data indicate that at least some mRNA is released from ribosomes during 5′‐translational blockage by arginine but not leucine codons, and they support proposals that premature termination of translation can occur in some conditions in vivo in the absence of a stop codon.
Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family
Authors: Roof W. D. , Fang H. Q. , Young K. D. , Sun Jianling , Young Ry
Published: November 2003

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis–trans peptidyl–prolyl isomerases (PPIases). slyD mutations affect plaque formation by the phage φX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of φX174 on these E R strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug‐independent phenotypes.
A developmentally regulated Streptomyces endoribonuclease resembles ribonuclease E of Escherichia coli
Authors: Hagège Juliette M. , Cohen Stanley N.
Published: November 2003

We report that the Streptomyces species S. lividans and S. coelicolor, morphologically complex Gram‐positive soil bacteria, contain a developmentally regulated endoribonuclease activity (here named RNase ES) that functionally and immunologically resembles Escherichia coli RNase E. In Streptomyces cells, RNA I — the antisense repressor of replication of ColE1‐type plasmids — is cleaved at sites attacked by RNase E. A Mg2+‐dependent endonuclease that produces RNase E‐like cleavages in RNA I and 9S ribosomal RNA was identified in S. lividans cell extracts. A Streptomyces peptide migrating at 70 kDa in SDS/polyacrylamide gels binds to RNase E substrates and reacts with three separate anti‐RNase E monoclonal antibodies; the endonucleolytic cleavage activity co‐purified with the immunoreactive 70 kDa peptide. We show that RNase ES activity is regulated during the Streptomyces life cycle: activity increased as cells progressed from exponential growth to stationary phase in liquid culture, or from mycelial growth to sporulation on solid media. While mutations that interfere with S. coelicolor development late in its life cycle did not prevent this developmentally associated increase in RNase ES activity, the increase was blocked by a mutation (bldA) that interferes early with both morphological and physiological differentiation.
The division during bacterial sporulation is symmetrically located in Sporosarcina ureae
Authors: Zhang Ling , Higgins Michael L. , Piggot Patrick J.
Published: November 2003

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae. Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spoIIA and cotE, of Bacillus subtilis were introduced into S. ureae by mobilization of plasmids containing the fusions from Escherichia coli. The fusions showed similar patterns of sporulation‐associated expression in S. ureae to those observed in B. subtilis. Formation of β‐galactosidase encoded by the spoIIA–lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spoIIA–lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli. It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.
Cascade regulation of the two CRP/FNR‐related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa
Authors: Arai Hiroyuki , Kodama Tohru , Igarashi Yasuo
Published: November 2003

ANR, an analogue of Escherichia coli FNR, has been shown to be necessary for denitrification, arginine deiminase activity and cyanide production of Pseudomonas aeruginosa. Another CRP/FNR‐related regulator, DNR, has also been shown to be necessary for denitrification. In this study, we have found that the transcription of the dnr gene is under the control of ANR. When the dnr gene was expressed by tac promoter in an anr mutant, the strain recovered the ability to grow under anaerobic conditions by denitrification. Nitrate, nitrite, nitric oxide and nitrous oxide reducing activities were expressed, and the structural genes for nitrite and nitric oxide reductases were transcribed under anaerobic conditions in the anr mutant strain transformed with the dnr gene. These findings suggest that the expression of the denitrification system is controlled not directly by ANR but indirectly via DNR. The arginine deiminase activity and cyanide production were not regulated by DNR.
Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA‐mediated assembly of a transcriptional activator at catabolic sites
Authors: Miller Coleen M. , Baumberg Simon , Stockley Peter G.
Published: November 2003

We have previously reported the initial characterization of a catabolic operator site (OrocA) for the Bacillus subtilis arginine repressor/activator protein AhrC. Here, we present the characterization by gel retardation and DNase I footprinting of both OrocA and a second catabolic operator site, OrocD. Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein. Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (Kd′  ≈ 90 nM for OrocA and ≈ 190 nM for OrocD). This difference may result from the lower match to the ARG box consensus of the OrocD site. Both catabolic operators show evidence for co‐operative binding with respect to protein concentration. Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B. subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t. This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross‐functioning of these proteins in vivo. The OrocA site is adjacent to a sharp, stable bend located 5′ to the catabolic operator. Circular permutation analysis has been used to determine the relative angle of bend (≈ 50°), its location and the effect of adding magnesium ions and/or AhrC protein. Protein binding increases the relative bend angle to ≈ 85°. Bending is shown to be associated with a number of A‐tracts in the upstream sequence. However, altering the phasing of the A‐tracts has little effect on the affinity for AhrC. Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity. Very surprisingly, the affinity of the OrocA site appears to increase in the presence of excess, specific competitor fragment, i.e. the system shows anti‐competitive effects. Competition is restored at high molar excesses of specific fragment over the protein. We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co‐operativity of binding and the anti‐competitive effects. These data suggest that the molecular interactions occurring between the prokaryotic arginine‐regulatory proteins and their operators may be more complex than is generally appreciated.
Cloning and characterization of a relA/spoT homologue from Bacillus subtilis
Authors: Wendrich Thomas M. , Marahiel Mohamed A.
Published: November 2003

A PCR‐amplified DNA fragment of the relA gene from genomic Bacillus subtilis DNA was used to isolate the entire relA/spoT homologue and two adjacent open reading frames (ORFs) from a λ ZAP Express library. The relA gene, which encodes a protein of 734 amino acid residues (aa), is flanked by an ORF (170 aa) that shares high similarity to adenine phosphoribosyltransferase genes (apt), and downstream by an ORF (131 aa) of unknown function. This genetic organization is similar to that in Streptomyces coelicolor A3(2) and Streptococcus equismilis H46A. relA shows significant similarity to the Escherichia coli relA and spoT genes, which are responsible for the synthesis and degradation of the highly phosphorylated guanosine nucleotides (p)ppGpp, triggering the stringent response. Deletion of the relA gene generated a (p)ppGpp0 phenotype that demonstrated its essential role in the response to amino acid deprivation and resulted in impaired/lowered induction of proteins involved in stress response as well as amino acid biosynthesis, as judged by two‐dimensional gel electrophoresis. The same effects of impaired induction of some σB‐independent proteins could also be shown in a sigB/relA double mutant, supporting the role of relA in derepression/induction of catabolic and anabolic genes during stringent response.
Glucose repression affects ion homeostasis in yeast through the regulation of the stress‐activated ENA1 gene
Authors: Alepuz Paula M. , Cunningham Kyle W. , Estruch Francisco
Published: November 2003

In this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.
Regulation of tryptophan biosynthesis: Trp‐ing the TRAP or how Bacillus subtilis reinvented the wheel
Authors: Babitzke Paul
Published: November 2003

The Bacillus subtilis tryptophan biosynthetic genes are regulated by TRAP. Radiographic crystallography indicates that the TRAP complex contains 11 identical subunits arranged in a doughnut‐like structure termed the β‐wheel. The trpEDCFBA operon is regulated by an attenuation mechanism in which tryptophan‐activated TRAP binds to 11 (G/U)AG repeats in the trp leader transcript. TRAP binding blocks formation of an anti‐terminator structure, thereby promoting the formation of an overlapping terminator, resulting in transcription termination preceding the structural genes. When TRAP is not activated, it is unable to bind to the transcript, which allows anti‐terminator formation and, hence, transcription of the operon. TRAP is also responsible for regulating translation of trpE and trpG. TRAP binding to trp operon readthrough transcripts promotes refolding of the RNA such that the trpE Shine–Dalgarno sequence is sequestered in a hairpin, thus inhibiting TrpE synthesis. In the case of trpG, TRAP binds to nine repeats that overlap the ribosome‐binding site, thereby blocking translation.
The ClpP protein, a subunit of the Clp protease, modulates ail gene expression in Yersinia enterocolitica
Authors: Pederson Kristin J. , Carlson Sharon , Pierson Dorothy E.
Published: November 2003

Yersinia enterocolitica is a gastrointestinal pathogen of humans and animals. Ail is a 17 kDa cell‐surface protein that confers on Y. enterocolitica resistance to serum killing and the ability to attach to and invade cells in vitro. The ail gene of Y. enterocolitica is regulated by temperature and growth phase. In stationary phase, ail transcript is only detected when bacteria are grown at the host temperature of 37°C. Our laboratory previously described a group of mini‐Tn10 mutants, which expressed ail in stationary phase at 28°C. In one of these mutants, DP5102::mini‐Tn10 3‐2, the mini‐Tn10 inserted into a gene encoding a protein with 90.3% identity to the ClpP protease subunit from Escherichia coli. Expression of ail in stationary phase at 28°C was also derepressed in a directed Y. enterocolitica clpP mutant. Analysis of ail transcripts in the wild‐type and clpP mutant strains indicated that there is a single start site of transcription of ail and that the effect of the clpP mutation was on the initiation of transcription at this site. Similar to E. coli, a clpX homologue was identified downstream of clpP. The Y. enterocolitica clpP gene complemented the clpP mutant phenotype, repressing the expression of both ail transcript levels and cell surface‐expressed Ail protein. Thus, ClpP has a role in the modulation of ail transcription in Y. enterocolitica.
Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non‐fermentable carbon sources
Authors: Jiang Feng , Rizavi Hooriyah S. , Greenberg Miriam L.
Published: November 2003

Cardiolipin is a unique dimeric phospholipid, which is present throughout the eukaryotic kingdom and is specifically localized in mitochondrial membranes. It is widely believed that mitochondria possess an essential requirement for this phospholipid. To determine whether cardiolipin is essential for yeast growth, we generated a cardiolipin synthase null mutant by disrupting the CLS1 gene (open reading frame YDL142c on chromosome IV) of Saccharomyces cerevisiae. Biochemical analysis of the mutant indicated that it had no cardiolipin synthase activity and no cardiolipin in its membranes. The enzyme phosphatidylglycerolphosphate synthase, which catalyses the committed step of the cardiolipin pathway, remained unaffected in the null mutant. Haploid cells containing the null allele are viable in media containing glucose, galactose or glycerol/ethanol as the sole carbon source, although growth in galactose or glycerol/ethanol is somewhat reduced in the mutant compared with the wild type. These results indicate that cardiolipin is not essential for the growth of S. cerevisiae in fermentable or non‐fermentable carbon sources.
DNA‐binding domain mutants of sigma‐N (σN, σ54) defective between closed and stable open promoter complex formation
Authors: Oguiza José A. , Buck Martin
Published: November 2003

The σN RNA polymerase binds promoters in a transcriptionally inactive form. Activation by enhancer binding positive control proteins results in the formation of an open promoter complex. In the closed complex, DNA sequences melted upon activation are close contacted by the σN C‐terminal DNA‐binding domain. Conserved phenylalanine residues within the DNA‐binding domain were mutated to examine their contribution to σN function. Mutants defective in supporting σN‐dependent growth and in vivo promoter activation were obtained. The mutant proteins were able to bind promoter DNA and to form an RNA polymerase holoenzyme closed complex in vitro. However, they were defective in response to activator in vitro. They failed in the formation of heparin‐stable promoter complexes characteristic of open promoter complexes. The σN mutant forms, displaying good promoter occupancy but poor open complex formation, appear defective for some function of the holoenzyme required after initial promoter recognition. The possibilities that the defect could be located in a DNA contact important for DNA melting or is associated with activator interaction and conformational change in σN are discussed.
The Rap and Hor proteins of Erwinia, Serratia and Yersinia: a novel subgroup in a growing superfamily of proteins regulating diverse physiological processes in bacterial pathogens
Authors: Thomson N. R. , Cox A. , Bycroft B. W. , Stewart G. S. A. B. , Williams P. , Salmond G. P. C.
Published: November 2003

The enteric bacterium Serratia marcescens is an opportunistic human pathogen. The strain ATCC39006 makes the red pigment, prodigiosin (Pig), and the β‐lactam antibiotic carbapenem (Car). Mutants were isolated that were concomitantly defective for Pig and Car production. These mutants were found to have a mutation in the rap gene (regulation of antibiotic and pigment). Sequence analysis of the rap gene revealed a predicted protein product showing strong homology to SlyA, originally thought to be a haemolytic virulence determinant in Salmonella typhimurium. Homologues of rap were detected in several bacterial genera, including Salmonella, Yersinia, Enterobacter, and species of the plant pathogen, Erwinia. The Erwinia horEr (homologue of rap) and the Yersinia horYe genes were also found to be very similar to rap and slyA. Marker exchange mutagenesis of horEr revealed that it encoded a regulatory protein controlling the production of antibiotic and exoenzyme virulence determinants in the phytopathogen, Erwinia carotovora subspecies carotovora. We have shown that these new homologues of SlyA form a highly conserved subgroup of a growing superfamily of bacterial regulatory proteins controlling diverse physiological processes in human, animal and plant pathogens.
Telomeres of the linear chromosomes of Lyme disease spirochaetes: nucleotide sequence and possible exchange with linear plasmid telomeres
Authors: Casjens Sherwood , Murphy Maria , DeLange Michael , Sampson Laura , Van Vugt René , Mun Huang Wai
Published: November 2003

Bacteria of the spirochaete genus Borrelia have linear chromosomes about 950 kbp in size. We report here that these linear chromosomes have covalently closed hairpin structures at their termini that are similar but not identical to those reported for linear plasmids carried by these organisms. Nucleotide sequence analysis of the chromosomal telomeric regions indicates that unique, apparently functional genes lie within a  few hundred bp of each of the telomeres, and that there is an imperfect 26 bp inverted repeat at the two telomeres. In addition, we characterize a major chromosomal length polymorphism within the right telomeric regions of various Borrelia isolates, and show that sequences similar to those near the right telomere are often found on linear plasmids in B. burgdorferi (sensu stricto) isolates from nature. Sequences similar to a number of other regions of the chromosome, including those near the left telomere, were not found on B. burgdorferi plasmids. These observations suggest that there has been historical exchange of genetic information between the linear plasmids and the right end of the linear chromosome.