bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection
Authors: Gérard Hervé C. , Krauße‐Opatz Birgit , Wang Zhao , Rudy Debbie , Rao J. Papa , Zeidler Henning , Schumacher H. Ralph , Whittum‐Hudson Judith A. , Köhler Lars , Hudson Alan P.
Published: March 2003

During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia‐associated arthritis. Hep‐2 cells were infected with K serovar C. trachomatis and harvested at t = 0–48 h post‐infection (p.i.; active). Human monocytes were infected similarly and harvested at t = 1–7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription–polymerase chain reaction (RT–PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep‐2 cells from 11 to 48 h p.i.; ftsK and ftsW, related to cell division, were expressed similarly. Real‐time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep‐2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1–7 p.i. and were weakly expressed in patient samples. Real‐time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.
Recombinational repair of chromosomal DNA double‐strand breaks generated by a restriction endonuclease
Authors: Cromie Gareth A. , Leach David R. F.
Published: March 2003

DNA double‐strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double‐strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double‐strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR ‘pathways’ are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild‐type cell. Our work suggests that the SbcCD–Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled‐coil linker, is specifically required for the co‐ordination of double‐strand break repair reactions in which two DNA ends are required to recombine at one target site.
In vitro transcription of PrfA‐dependent and  ‐independent genes of Listeria monocytogenes
Authors: Lalic‐Mülthaler M. , Bohne J. , Goebel W.
Published: March 2003

In vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied. Whereas transcription from Phly, PplcA and PactA is strictly PrfA‐dependent, that from Piap, PprfA1/2 and, unexpectedly, also from Pmpl is independent. Initiation of in vitro transcription at all tested promoters except PprfA requires high concentrations of ATP but not GTP. The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript. RNA polymerase prepared from L. monocytogenes cultured either in rich culture medium (RNAPBHI), exposed to heat shock conditions (RNAP48) or conditioned in minimal essential medium (RNAPMEM) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters. Transcription starting from the PrfA‐dependent promoters PactA and Phly is enhanced with RNAP48 and RNAPMEM (in relation to Piap–mediated transcription), while transcription from the other promoters is reduced when compared with RNAPBHI. These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor.
Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147‐2
Authors: Monds Russell D. , Silby Mark W. , Mahanty H. Khris
Published: March 2003

We report the isolation of insertional mutations to the pstC and pstA genes of the phosphate‐specific transport (pst) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147‐2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (Pi) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular Pi. The two‐component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to Pi‐limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in Pi‐limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low‐Pi environments, and that phoR mutations uncouple PA147‐2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non‐limiting Pi environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147‐2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to Pi assimilation and Pho regulon control in P. aureofaciens.
Evidence that F‐plasmid proteins TraV, TraK and TraB assemble into an envelope‐spanning structure in Escherichia coli
Authors: Harris Robin L. , Hombs Veronica , Silverman Philip M.
Published: March 2003

We have examined the role of the F‐plasmid TraV outer membrane lipoprotein in the assembly of F‐pili. Yeast two‐hybrid analysis with a traV bait repeatedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclusively identified traK preys. Several additional observations support the hypothesis that TraV, TraK and TraB form a complex in Escherichia coli that spans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two‐hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bridges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra+ cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV–TraK–TraB complex anchored to the outer membrane via the TraV lipoprotein. Further, in traK mutant cells, TraV failed to accumulate to a detectable level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to its normal level. Both TraK and TraV accumulated in traB2[Am] cells; however, the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK‐binding domain suggested by yeast two‐hybrid analysis. We propose that TraV is the outer membrane anchor for a trans‐envelope, Tra protein structure required for the assembly of F‐pili and possibly for other events of conjugal DNA transfer.
Functional analysis of the Bacillus subtilis morphogenetic spore coat protein CotE
Authors: Little Shawn , Driks Adam
Published: March 2003

Bacterial spores are surrounded by a multilayered proteinaceous shell called the coat. In Bacillus subtilis, a coat protein called CotE guides the assembly of a major subset of coat proteins. To understand how CotE carries out its role in coat morphogenesis, we subjected its gene to mutagenesis and studied the effects of altered versions of CotE on coat formation. We identified regions within the C‐terminal 28 amino acids that direct the deposition of the coat proteins CotA, CotB, CotG, CotSA, CotS and 35 kDa and 49 kDa proteins likely to be the spore proteins CotR (formerly known as YvdO) and YaaH respectively. The timing and genetic dependency of CotR accumulation are consistent with control of its gene by σK and GerE. In addition, we identified a 35‐amino‐acid internal region involved in targeting of CotE to the forespore. Finally, we found that sequences within this 35‐amino‐acid region as well as within an 18‐amino‐acid stretch in the N‐terminus of CotE direct the formation of CotE multimers, most probably homooligomers. These results suggest that: (i) most interactions between CotE and the coat proteins assembled under CotE control take place at the CotE C‐terminus; (ii) an internal region of CotE connects it with the forespore surface; and (iii) interactions between CotE molecules depend on residues within an 18‐amino‐acid region in the N‐terminal half of CotE.
Interactions of the Trichoderma reesei rho3 with the secretory pathway in yeast and T. reesei
Authors: Vasara Tuija , Salusjärvi Laura , Raudaskoski Marjatta , Keränen Sirkka , Penttilä Merja , Saloheimo Markku
Published: March 2003

We recently isolated from the filamentous fungus Trichoderma reesei (Hypocrea jecorina) a gene encoding RHOIII as a multicopy suppressor of the yeast temperature‐sensitive secretory mutation, sec15‐1. To characterize this gene further, we tested its ability to suppress other late‐acting secretory mutations. The growth defect of yeast strains with sec1‐1, sec1‐11, sec3‐2, sec6‐4 and sec8‐9 mutations was suppressed. Expression of rho3 also improved the impaired actin organization of sec15‐1 cells at +38°C. Overproduction of yeast Rho3p using the same expression vector as T. reesei RHOIII appeared to be toxic in sec3‐101, sec5‐24, sec8‐9, sec10‐2 and sec15‐1 cells. When expressed from the GAL1 promoter, RHO3 suppressed the growth defect of sec1 at the restrictive temperature and inhibited the growth of sec3‐101 at the permissive temperature. Disruption of the rho3 gene in the T. reesei genome did not affect the hyphal or colony morphology nor the cellular cytoskeleton organization. Furthermore, the growth of T. reesei was not affected on glucose by the rho3 disruption. Instead, both growth and protein secretion of T. reesei in cellulose cultures was remarkably decreased in rho3 disruptant strains when compared with the parental strain. These results suggest that rho3 is involved in secretion processes in T. reesei.
The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli
Authors: Godfrin‐Estevenon Anne‐Marie , Pasta Franck , Lane David
Published: March 2003

The bacteria for which there is evidence that proteins of the ParAB family act in chromosome segregation also undergo developmental transitions that involve the ParAB homologues, raising the question of whether the partition activity is equivalent to that of plasmid partition systems. We have investigated the role in partition of the parAB locus of a free‐living bacterium, Pseudomonas putida, not known to pass through developmental phases. A parAB deletion mutant, compared with wild type, showed slightly higher frequencies of anucleate cells in exponen‐tially growing cultures but much higher frequencies in deceleration phase. This increase was growth medium dependent. Oversupply of ParA and ParB proteins also raised anucleate cell levels, specifically in the deceleration phase, in wild‐type and mutant strains and regardless of medium, as well as generating abnormal cell morphologies. Absence or oversupply of ParAB function had either slight or considerable effects on growth rate, depending on temperature and medium. The need for the Par proteins in chromosome partition thus appears to be subject to the cell’s physiological state. Three sequences similar to cis‐acting stabilization sites of Bacillus subtilis are present in the P. putida oriC–parAB region. One was inserted into an unstable mini‐F and shown to stabilize it in E. coli in a ParAB‐dependent manner.
Identification of binding sites for the group A streptococcal global regulator CovR
Authors: Federle Michael J. , Scott June R.
Published: March 2003

The CovRS two‐component system (also called CsrRS) of the group A streptococcus (GAS) acts as a global regulator, influencing the transcription of at least six virulence factors. The synthesis of the hyaluronic acid capsule, a virulence factor encoded by the hasABC operon, is negatively regulated by CovRS. We confirmed that

phosphorylation of CovR increases its binding to a DNA fragment containing the hasA

promoter. Using DNase I footprinting, we identified five binding sites surrounding

the hasA promoter from bases –79 to +73 (where +1 is

the start of transcription). One pair of thymines within each binding site appears

to be necessary for CovR binding in vitro, as shown by uracil interference

analysis. When each of these thymine pairs was altered by site‐directed mutagenesis,

CovR binding was reduced in vitro, confirming the role of each thymine pair

in binding. Using a transcriptional reporter system with a single chromosomal copy

of PhasA–gusA, we demonstrated the importance of each of four of these

binding sites for CovR repression of the hasA promoter. Based on this information, we propose a consensus sequence for CovR binding to DNA.
The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K‐12: two nitrate and three nitrite transporters
Authors: Clegg Stephanie , Yu Feng , Griffiths Lesley , Cole Jeffrey A.
Published: March 2003

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria

is unknown. We report the construction and use of four isogenic strains that lack

nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations

of narK and narU. The active site of the only functional nitrate reductase,

nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four

strains is totally dependent upon a mechanism for importing nitrate. These strains

were exploited to determine the roles of NarK and NarU in both nitrate and nitrite

transport. Single mutants that lack either NarK or NarU were competent for nitrate‐dependent

anaerobic growth on a non‐fermentable carbon source, glycerol. They transported and

reduced nitrate almost as rapidly as the parental strain. In contrast, the narK–narU

double mutant was defective in nitrate‐dependent growth unless nitrate transport

was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also

unable to catalyse nitrate reduction in the presence of physiological electron donors.

Synthesis of active nitrate reductase A and the cytoplasmic, NADH‐dependent nitrite

reductase were unaffected by the narK and narU mutations. The rate

of nitrite reduction catalysed by the cytoplasmic, NADH‐dependent nitrite reductase

by the double mutant was almost as rapid as that of the NarK+‐NarU+

strain, indicating that there is a mechanism for nitrite uptake by E. coli

that is in‐dependent of either NarK or NarU. The nir operon encodes a soluble,

cytoplasmic nitrite reductase that catalyses NADH‐dependent reduction of nitrite

to ammonia. One additional component that contributes to nitrite uptake was shown

to be NirC, the hydrophobic product of the third gene of the nir operon, which

is predicted to be a polytopic membrane protein with six membrane‐spanning helices.

Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal

rate that was fully restored by a single chromosomal copy of either narK or

nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex‐cretion, not nitrite uptake.
Parallel pathways for oxidation of 14‐membered polyketide macrolactones in Saccharopolyspora erythraea
Authors: Gaisser Sabine , Lill Rachel , Staunton James , Méndez Carmen , Salas José , Leadlay Peter F.
Published: March 2003

The glycosyltransferases OleG1 and OleG2 and the cytochrome P450 oxidase OleP from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus have
been expressed, either separately or from artificial gene cassettes, in strains of
Saccharopolyspora erythraea blocked in erythromycin biosynthesis, to investigate
their potential for the production of diverse novel macrolides from erythronolide
precursors. OleP was found to oxidize 6‐deoxyerythronolide B, but not erythronolide
B. However, OleP did oxidize derivatives of erythronolide B in which a neutral sugar
is attached at C‐3. The oxidized products 3‐O‐mycarosyl‐8a‐hydroxyerythronolide
B, 3‐O‐mycarosyl‐8,8a‐epoxyerythronolide B, 6‐deoxy‐8‐hydroxyerythronolide
B and the olefin 6‐deoxy‐8,8a‐dehydroerythronolide B were all isolated and their
structures determined. When oleP and the mycarosyltransferase eryBV
were co‐expressed in a gene cassette, 3‐O‐mycarosyl‐6‐deoxy‐8,8a‐dihydroxyerythronolide
B was directly obtained. When oleG2 was co‐expressed in a gene cassette together
with oleP, 6‐deoxyerythronolide B was converted into a mixture of 3‐O‐rhamnosyl‐6‐deoxy‐8,8a‐dehydroerythronolide
B and 3‐O‐rhamnosyl‐6‐deoxy‐8,8a‐dihydroxyerythronolide B, confirming previous
reports that OleG2 can transfer rhamnose, and confirming that oxidation by OleP and
attachment of the neutral sugar to the aglycone can occur in either order. Similarly,
four different 3‐O‐mycarosylerythronolides were found to be substrates for the desosaminyltransferase OleG1. These results provide additional insight into the nature of the intermediates in OleP‐mediated oxidation, and suggest that oleandomycin biosynthesis might follow parallel pathways in which epoxidation either precedes or follows attachment of the neutral sugar.
Spx (YjbD), a negative effector of competence in Bacillus subtilis, enhances ClpC–MecA–ComK interaction
Authors: Nakano Michiko M. , Nakano Shunji , Zuber Peter
Published: March 2003

ComK, a key transcriptional regulator in the development of competence in Bacillus subtilis, is required for its own transcription as well as that of the late competence genes encoding proteins involved in DNA uptake. ComK is sequestered in a complex with ClpC and MecA until a peptide, ComS, accumulates in cells. ComS releases ComK from the inhibitory complex, thus allowing ComK to carry out its function as a transcriptional activator. Spx (formerly YjbD), a negative effector of competence, accumulates in clpP mutants. High concentrations of Spx may be responsible for the inability of clpP mutants to become competent because spx mutations are able to restore competence in the clpP mutant. In this paper, we showed, based on in vitro experiments, that Spx forms a quaternary complex with ClpC, MecA and ComK and enhances ComK binding to ClpC–MecA. Two ComS alanine substitution mutants (I13A and W43A), previously shown to be defective in vivo, were less efficient in releasing ComK from ClpC–MecA. The I13A mutant with a weaker binding affinity to MecA was inefficient in releasing ComK regardless of whether Spx was present. In contrast, the defect of the W43A mutant in dissociating ComK was more readily observed in the presence of Spx. Spx is a highly conserved protein among Gram‐positive bacteria, in which it may function closely with the protease adaptor protein, MecA.
Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin‐containing enzymes
Authors: McDevitt Christopher A. , Hugenholtz Philip , Hanson Graeme R. , McEwan Alastair G.
Published: March 2003

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop‐terin‐containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron–sulphur clusters. Analysis of the N‐terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec‐dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo‐trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.
Cyclin‐dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii
Authors: Khan Farzana , Tang Jianzhong , Qin Chang‐le , Kim Kami
Published: March 2003

The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin‐dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2‐HA‐wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2‐HA‐wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2‐HA‐wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant‐negative mutant of TPK2 (TPK2‐HA‐dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2‐HA‐dn revealed an increase in the fraction of cells in S‐phase when compared with TPK2‐HA‐wt transfected parasites. Expression of TPK2‐HA‐wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.
Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced γ‐butyrolactone receptor
Authors: Stratigopoulos George , Gandecha Atul R. , Cundliffe Eric
Published: March 2003

During promoter‐probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP re‐sulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.
Adaptation of protein secretion to extremely high‐salt conditions by extensive use of the twin‐arginine translocation pathway
Authors: Rose R. Wesley , Brüser Thomas , Kissinger Jessica C. , Pohlschröder Mechthild
Published: March 2003

Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such ‘hostile’ conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non‐haloarchaea, and most probably represents an adaptation to the high‐salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin‐arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole‐genome analysis of Halobacterium sp. NRC‐1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non‐redox proteins, the homologues of which in non‐haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin‐arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high‐salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.
Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii
Authors: Maisnier‐Patin Sophie , Malandrin Laurence , Birkeland Nils‐Kåre , Bernander Rolf
Published: March 2003

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.
Suppression of a nuclear frameshift mutation by a mitochondrial tRNA in the yeast Kluyveromyces lactis‡
Authors: Fiori Alessandro , Francisci Silvia , Falcone Claudio
Published: March 2003

A fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine‐tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory‐deficient mutant of this yeast. The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al. 2000 Yeast 16: 307–314). We report here that the mitochondrial tRNAVAL gene, when transformed into K. lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA. In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt‐tRNAVAL from the nucleus. Suppression could occur through a non‐canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation. This is a new case of informational suppression in that the suppression of a chromosomal mutation is not casused by a second mutation but to a mislocalization/expression of a mt‐tRNA.
DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA
Authors: Davis Elaine O. , Springer Burkhard , Gopaul Krishna K. , Papavinasasundaram K. G. , Sander Peter , Böttger Erik C.
Published: March 2003

The ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA.
Saccharomyces cerevisiae C1D is implicated in both non‐homologous DNA end joining and homologous recombination
Authors: Erdemir Tuba , Bilican Bilada , Cagatay Tolga , Goding Colin R. , Yavuzer Ugur
Published: March 2003

C1D is a gamma‐irradiation inducible nuclear matrix protein that interacts with and activates the DNA‐dependent protein kinase (DNA‐PK) that is essential for the repair of the DNA double‐strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break‐point junctions, and that over expression of C1D can induce p53‐dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non‐homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid‐based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double‐strand breaks.
The Neisseria meningitidis adhesion regulatory protein CrgA acts through oligomerization and interaction with RNA polymerase
Authors: Deghmane Ala‐Eddine , Taha Muhamed‐Kheir
Published: March 2003

CrgA is a LysR‐type transcriptional regulator involved in the intimate adhesion of Neisseria meningitidis to target human epithelial cells. It is negatively autoregulated, and its expression is transiently induced upon contact with target cells. We analysed the functional organization of CrgA using in frame deleted proteins. Four truncated proteins were constructed and purified. They were deleted between residues 20 and 40, 121 and 154, 111 and 181 and 268 and 291. Meningococcal mutants harbouring the corresponding deleted crgA alleles were also constructed. All mutants showed a reduced ability to adhere to epithelial cells. β‐Galactosidase activity assays using a crgA–lacZ transcriptional fusion showed that all the mutations except the 268–291 deletion resulted in loss of induction upon contact with target cells. Gel mobility shift assays and cross‐linking assays showed that the oligomerization of CrgA is required for DNA binding and that the N‐terminal part of CrgA is directly involved in DNA binding through a helix–turn–helix motif. The C‐terminal region is also involved in DNA binding, probably by permitting the oligomerization of CrgA. The C‐terminal region also seemed to interact with RNA polymerase. Therefore, the binding of CrgA and its interaction with RNA polymerase may inhibit the clearance of meningococcal promoters that are repressed by CrgA during the intimate adhesion of N. meningitidis to target cells.
Localization of Mycoplasma pneumoniae cytadherence‐associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure
Authors: Balish Mitchell F. , Santurri Ryan T. , Ricci Alessondra M. , Lee Kyungok K. , Krause Duncan C.
Published: March 2003

The terminal organelle of the cell wall‐less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane‐bound extension of the mycoplasma cell possesses a characteristic electron‐dense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence‐accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody‐based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild‐type M. pneumoniae and the hmw2– mutant I‐2. The fusion protein was produced in both backgrounds at wild‐type levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I‐2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electron‐dense core of the terminal organelle.
Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum‐sensing‐dependent and ‐independent pathways
Authors: Slater Holly , Crow Matthew , Everson Lee , Salmond George P. C.
Published: March 2003

Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1‐carbapen‐2‐em‐3‐carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N‐acyl homoserine lactone (N‐AHL) quorum sensing, with synthesis of N‐AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum‐sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA–O, are transcribed as a single polycistronic message in an N‐AHL‐dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR‐type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N‐AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone‐independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild‐type backgrounds confers a hyper‐Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high‐affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper‐production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006.
Characterization of a novel inhibitory feedback of the anti‐anti‐sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis
Authors: Arabolaza Ana L. , Nakamura Akira , Pedrido María E. , Martelotto Luciano , Orsaria Lelia , Grau Roberto R.
Published: March 2003

Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell‐specific activation of the transcription factors σF and σE. Synthesis of these σ factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti‐anti‐σF SpoIIAA as inhibitor of Spo0A activation. This effect did not require σF activity, and it was abolished by expression of the phosphorelay‐independent form Spo0A‐Sad67 indicating that SpoIIAA directly interfered with Spo0A∼P generation. IPTG‐directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA‐P. Furthermore, expression of the non‐phosphorylatable mutant SpoIIAAS58A (SpoIIAA‐like), but not SpoIIAAS58D (SpoIIAA‐P‐like), completely blocked Spo0A‐dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of σF and σE during development.
A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain
Authors: Oliver David C. , Huang George , Nodel Elena , Pleasance Steve , Fernandez Rachel C.
Published: March 2003

Autotransporter secretion represents a unique mechanism that Gram‐negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa α‐domain and a 30 kDa β domain. The α domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the β domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the β‐domain to ferry the passenger domain to the bacterial surface through a channel formed by the β‐domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601–Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT‐deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601–Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.
Tn5 as a model for understanding DNA transposition
Authors: Reznikoff William S.
Published: March 2003

Tn5 is an excellent model system for understanding the molecular basis of DNA‐mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild‐type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.
Anorectal functions in patients with spinal cord injury
Authors: GREVING  , TEGENTHOFF  , NEDJAT  , ORTH  , BÖTEL  , MEISTER  , MICKLEFIELD  , MAY  , ENCK
Published: March 2003

We wished to establish anorectal functions in patients with spinal cord lesions, related to the level of lesion and its completeness. We also wished to determine the value of neurophysiological tests for completeness of transsections in comparison with manometry and visceral sensory testing.

In 32 patients (31.5 ± 14.1 years, 25 males) with spinal trauma, completeness of transsection was assessed clinically. In 16 of these patients (30 ± 15.6 years, nine males), a neurological work‐up included recording of somatosensory evoked potentials (SEP) and motor evoked potentials (MEP) from the pudendal nerve within the first week after trauma. Also, anal sphincter EMG and pudendal nerve terminal motor latency (PNTML) were assessed. All patients also underwent conventional anorectal manometry and visceral sensory testing.

Of all 32 patients, 15 were judged as `complete' based on their clinical signs. Of those 16 tested neurologically, seven were labelled `complete' since no MEP or SEP were detectable; one had pudendal SEP and MEP present, while SEP were present but delayed (47.0 ± 8.8 msec) in the remaining patients. In four of these patients, also MEP were recorded (27.9 ± 5.2 msec) and normal. PNTML was present in 12/16 patients independent of the completeness of lesion, and was rated normal in nine and delayed in three patients. EMG was normal in five, and pathological in 11 cases. In 5/15 cases of those judged as `complete' (in 3/7 evaluated neurologically), visceral sensory testing revealed a minimal threshold for rectal perception of distension of 44 mL (range: 10–130), which sometimes was also perceived as urge to defecate. In a further case, manometry showed major voluntary action of the anal sphincter. These patients had lesions at all levels of the spinal column, ranging from cervical (C4,C6,C7) via thoratical (2 × T7,T8,T12) to lumbar segments.

Anorectal function testing, and specifically visceral sensory testing may be superior to neurological assessment of `completeness' of spinal cord lesions. It may be that visceral afferent pathways others than spinothalamic tract are involved in rectal perception that are less accessible to conventional neurophysiological diagnostic work‐up.
Hyperthermia prevents functional, histological and biochemical abnormalities induced during ileitis
Authors: GOLDHILL  , STOJADINOVIC  , KIANG  , SMALLRIDGE  , DONOHUE
Published: March 2003

Inflammatory bowel disease is associated with altered intestinal motility and epithelial damage. Hyperthermia induces heat shock protein expression, components of a basic cellular defence mechanism, and consequently prevents ischaemic damage. Here we investigate whether hyperthermia may prevent altered smooth muscle function as well as underlying inflammation in a model of inflammatory bowel disease. Ileal heat shock protein expression was induced in rats by hyperthermic shock (41.5 °C; 5 min). Two hours after heating or sham treatment, ileitis was evoked by TNBS. Ileal samples were taken 4 h later to determine the contractile response of circular muscle strips, and to measure heat shock protein expression, LTB4 generation and damage/inflammation. Ileitis was associated with an increase in the contractile response of circular muscle to substance P but not neurokinin A or nerve stimulation. Hyperthermia induced heat shock protein expression and also prevented this functional change as well as TNBS‐induced LTB4 production, subsequent infiltration of neutrophils and epithelial damage. Thus, intestinal inflammation is associated with alterations in tachykinergic control of smooth muscle as well as inflammatory changes. Hyperthermia prevents these changes and induces heat shock protein expression. Pharmacological induction of these proteins may offer a novel clinical strategy in treating both of these aspects of disease.