bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence–function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli
Authors: Dassa Elie
Published: October 2006

The maIG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy‐dependent transport of maltose and maltodextrins in Escherichia coli. The MalG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MalG subunit. Fifteen mutations have been isolated in malG by random linker insertion mutagenesis. Two regions essential for maltose transport have been identified. In particular, a hydro philic region containing the peptidic motif EAA—G———I‐LP, highly conserved among inner membrane proteins from binding protein‐dependent transport systems, is essential for maltose transport.

The results also show that several regions of MalG are not essential for function. A region (residues 30–50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MalG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein. A region located at the middle of the protein (residues 153–157) is not essential for the function of the protein. A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C‐terminus of the protein.
The strongly conserved carboxyl‐terminus glycine‐methionine motif of the Escherichia coli GroEL chaperonin is dispensable
Authors: McLennan N. F. , Girshovich A. S. , Lissin N. M. , Charters Y. , Masters M.
Published: October 2006

The universally distributed heat‐shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70S, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild‐type protein can suppress a number of temperature‐sensitive mutations; the truncated protein lacks this ability.
MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins
Authors: Allaoui Abdelmounaaïm , Sansonetti Philippe J. , Parsot Claude
Published: October 2006

The invasive phenotype of Shigella flexneri is conferred by a 220 kb virulence plasmid, pWR100, that encodes both the Ipa proteins, which are involved in the entry process, and factors which are required for the export and correct localization of the Ipa proteins. We have characterized the mxiD gene, whose expression, like that of the ipa operon, is regulated by temperature. After inactivation of mxiD, the mutant strain was unable to invade HeLa cells and to provoke keratoconjunctivitis in guinea‐pigs. Analysis of culture supernatants indicated that wild‐type S. flexneri secretes about nine polypeptides and that secretion of several of these, including IpaA, IpaB, and IpaC, is abolished in the mxiD mutant. Examination of the membrane proteins of the wild‐type and mxiD strains suggested that MxiD is an outer membrane protein. Amino acid sequence comparison revealed that MxiD is homologous to the YscC protein of Yersinia enterocolitica and to the C‐terminal region of the PulD protein of Klebsiella pneumoniae. Both YscC and PulD are involved in extracellular protein secretion. These results indicate that MxiD is an essential component of the Ipa secretion apparatus.
The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP‐dependent translocators
Authors: Karow Margaret , Georgopoulos Costa
Published: October 2006

We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature‐sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and encodes a protein with an estimated molecular mass of 64460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP‐dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.
C‐terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss‐of‐function and gain‐of‐function phenotypes
Authors: Stankovich M. , Platt A. , Caddick M. X. , Langdon T. , Shaffer P. M. , Arst H. N.
Published: October 2006

Mutations truncating as many as 143 C‐terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain‐of‐function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C‐terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C‐terminal residues, virtually all amino acids C‐terminal to the DNA‐binding region, is compatible with retention of some areA function.
Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells
Authors: Altmeyer Ralf M. , McNern John K. , Bossio J. C. , Rosenshine Ilan , Finlay B. Brett , Galán Jorge E.
Published: October 2006

Our laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host‐adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147‐amino‐acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of ∼16000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.
A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage
Authors: Switalski Lech M. , Patti Joseph M. , Butcher Wade , Gristina Anthony G. , Speziale Pietro , Höök Magnus
Published: October 2006

Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187‐amino‐acid repeat motif. Collagen receptor‐positive strains adhered to both collagen substrata and cartilage in a time‐dependent process. Collagen receptor‐specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.
Signal transduction between the two regulatory components involved in the regulation of the kdpABC operon in Escherichia coli: Phosphorylation‐dependent functioning of the positive regulator, KdpE
Authors: Nakashima K. , Sugiura A. , Kanamaru K. , Mizuno T.
Published: October 2006

The proteins KdpD and KdpE are crucial to the osmotic regulation of the kdpABC operon that is responsible for the high‐affinity K+ ion transport system in Escherichia coli. We demonstrated previously that the response regulator, KdpE, is capable of undergoing Phosphorylation mediated by the sensory protein kinase, KdpD. In this study, we obtained biochemical evidence supporting the view that when KdpE is phosphorylated, it takes on an active form that exhibits relatively high affinity for the kdpABC promoter, which in turn results in activation of the kdpABC operon. It was also suggested that the central hydrophobic domain of KdpD, which is conceivably responsible for membrane anchoring of this protein, plays a role in the signalling mechanism underlying KdpE Phosphorylation in response to hyperosmotic stress.
Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric‐pseudobactin transport
Authors: Bitter Wilbert , Tommassen Jan , Weisbeek Peter J.
Published: October 2006

Catechol‐cephalosporins are siderophore‐like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric‐siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron‐limiting conditions, and could not utilize different ferric‐siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N‐terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy‐coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB‐like energy‐transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.
Topology of PhoE porin: the ‘eyelet’ region
Authors: Struyvé Marlies , Visser Jan , Adriaanse Henriëtte , Benz Roland , Tommassen Jan
Published: October 2006

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic β‐sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R capsulatus porin, which has an ‘eyelet’ region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar ‘eyelet’ region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot‐and‐mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single‐channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.
Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases
Authors: Wandersman Cécile , Létoffé Sylvie
Published: October 2006

The presence of the α‐haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram‐negative envelope (owing to its large size) (Mr 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non‐haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced α‐haemolysin secretion as weli as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.
Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL
Authors: Tyson K. L. , Bell A. I. , Cole J. A. , Busby S. J. W.
Published: October 2006

Transcription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions −52 and −30 upstream of the transcription startsite. The 20 base pairs from position −79 to −60 contain an inverted repeat of two 10‐base sequence elements that are related to sequences at the NarL‐binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL‐binding sequence. Mutations in the putative NarL‐binding site at the nirB promoter decrease FNR‐dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry‐related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate‐responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry‐related positions in the NarL‐binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.
Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae
Authors: Eiglmeier K. , Honoré N. , Woods S. A. , Caudron B. , Cole S. T.
Published: October 2006

In an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.B Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.
Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif‐Str regions
Authors: Honoré N. , Bergh S. , Chanteau S. , Doucet‐Populaire F. , Eiglmeir K. , Garnier T. , Georges C. , Launois P. , Limpaiboon T. , Newton S. , Niang K. , del Portillo P. , Ramesh G. R. , Reddi P. , Ridel P. R. , Sittisombut N. , Wu‐Hunte S. , Cole S. T.
Published: October 2006

The nucleotide sequence of cosmid B1790, carrying the Rif‐Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β'subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so‐called ABC family of ATP‐binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.
The general amino acid control regulates MET4, which encodes a methionine‐pathway‐specific transcriptional activator of Saccharomyces cerevisiae
Authors: Mountain Harry A. , Byström Anders S. , Korch Christopher
Published: October 2006

A met4 mutant of Saccharomyces cerevisiae was unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway. The sequence of the cloned MET4 gene allowed the previously sequenced flanking LEU4 and POL1 genes to be linked to MET4 into a 10 327 bp contiguous region of chromosome XIV. From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C‐terminus, preceded by both acidic and basic regions. Thus, MET4 belongs to the family of basic leucine zipper trans‐activator proteins. Disruption of MET4 resulted in methionine auxotrophy with no other phenotype. Transcriptional studies showed that MET4 was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4. GCN4 binding sequences are present between the divergently transcribed MET4 and LEU4 genes. Over‐expression of MET4 resulted in leaky expression from the otherwise tightly regulated MET3 promoter under its control. The presence of consensus sequences for other potential regulatory elements in the MET4 promoter suggests a complex regulation of this gene.
Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin
Authors: Kukkonen Maini , Raunio Tiina , Virkola Ritva , Lähteenmäki Kaarina , Mäkelä P. Helena , Klemm Per , Clegg Steven , Korhonen Timo K.
Published: October 2006

Adherence of type‐1‐fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type‐1‐fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non‐fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside‐inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae‐coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type‐1 ‐fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type‐1‐fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type‐1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type‐1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.
Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains
Authors: Viret Jean‐François , Cryz Stanley J. , Lang Alois B. , Favre Didier
Published: October 2006

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype‐specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K‐12 and RI types, but neither to Salmonella (Ra‐type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1‐type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfdsonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri‐type LPS. Co‐expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase‐l‐like S. sonnei LPS in E. coli K‐12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.
Effects of tRNALeu1 overproduction in Escherichia coli
Authors: Wahab Samir Z. , Rowley Karla O. , Holmes W. Michael
Published: October 2006

Strains of Escherichia coli have been produced which express very high levels of the tRNAleu1 isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNALeu1 gene. Most transformants grew very slowly and exhibited a 15‐fold increase in cellular concentrations of tRNALeu1 As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNALeu1. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two‐ and threefold. Analyses of leucyl tRNA isolated from these slow‐growing strains showed that at least 90% of the detectable tRNALeu1 was hypomodified as judged by altered mobility on RPC‐5 reverse‐phase columns, and by specific modification assays using tRNA(m1G)‐methyltransferase and pseudo‐uridylate synthetase. Analysis of fast‐growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild‐type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNALeu1 operon under the control of the T7 promoter was prepared which, when induced, produced six‐ to sevenfold increases in tRNALeu1 levels. This level of tRNALeu1 titrated the modification system as judged by RPC‐5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein‐synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.
Mutagenesis and functional analysis of the Escherichia coli tRNALeu1 promoter
Authors: Bauer Brian F. , Elford Roberta M. , Holmes W. Michael
Published: October 2006

The leuV promoter which produces tRNALeu1 in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth‐rate‐dependent regulation (GDR). Each mutant promoter was ligated with a β‐galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the −10 consensus region, when altered, caused remarkable up‐promoter effects, increasing efficiency in some cases almost 10‐fold. One up mutation which had five successive T residues in the‘discriminator’region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the −35 region to bring it to consensus increased promoter strength 18‐fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR‐disturbed. AM upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.
Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate
Authors: Tubulekas Ioannis , Hughes Diarmaid
Published: October 2006

We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF‐Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl‐tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild‐type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is Incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.
Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro
Authors: Ruehl W. W. , Marrs C. , Beard M. K. , Shokooki V. , Hinojoza J. R. , Banks S. , Bieber D. , Mattick J. S.
Published: October 2006

Moraxella bovis, the causative agent of infectious bovine keratoconjunctivttis, exhibits several virulence factors, including pili, haemotysin, leukotoxin, and proteases. The pili are filamentous appendages which mediate bacterial adherence. Prior studies have shown that Q‐piliated M. bovis Epp63 are more infectious and more pathogenic than l‐piliated and nonpiliated isogenic variants, suggesting that Q pili perse or traits associated with Q‐pilin expression, promote the early association of Q‐pillated bacteria with bovine corneal tissue. In order to better evaluate the role of Q pili in M. bovis attachment, several M. bovis strains and a recombinant P. aeruginosa strain which elaborates M. bovis Q pili but not P. aeruginosa PAK pili, were evaluated using an in vitro corneal attachment assay. For each strain tested, piliated organisms attached better than non‐piliated bacteria. M. bovis Epp63 Q‐piIiated bacteria adhered better than either the l‐piliated or non‐piliated isogenic variants. Finally, recombinant P. aeruginosa organisms elaborating M. bovis Q pili adhered better than the parent P. aeruginosa strain which did not produce M. bovis pili. These results indicate that the presence of pili, especially Q pili, enhances the attachment of bacteria to bovine cornea In vitro.
The origin of greater‐than‐unit‐length plasmids generated during bacterial conjugation
Authors: Erickson Matthew J. , Meyer Richard J.
Published: October 2006

In Gram‐negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid DNA strand with 5′ to 3′ polarity. Transfer is initiated by nicking of the duplex DNA at a particular site, i.e. the origin of transfer (oriT). We constructed plasmids containing two directly repeated copies of oriT, derived from the broad‐host‐range plasmid R1162 and flanking the lac operator. The number of lacO copies in the plasmid after transfer could be determined from the colour of transconjugant colonies on medium containing X‐Gal. When the oriTs were mutated to prevent initiation and termination of transfer at the same oriTs, almost all of the transconjugant cells contained greater‐than‐unit‐length plasmids with two copies of lacO and three copies of oriT. We show that these molecules were generated by an intramolecular, conjugation dependent mechanism unlikely to depend solely on a pre‐existing population of circular or linear multimers in donor cells. We propose that the greater‐than‐unit‐length molecules were instead generated by a rolling‐circle mechanism of DNA replication.
Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence
Authors: Arroyo Rossana , González‐Robles Arturo , Martínez‐Palomo Adolfo , Alderete J. F.
Published: October 2006

The cytoadherence of Trichomonas vaginalis, the sexually transmitted flagellated protozoan, to vaginal epithelial cells (VECs) is the key to infection. Electron microscopy revealed that in vitro‐grown parasites having typical globular shape transformed rapidly after contact with VECs into thin, flat, amoeboid cells, maximizing the area of adhesion to the surface of VECs. Amoebic trichomonads formed filopodia and pseudopodia, which interdigitated at distinct sites on the plasma membrane of target cells. In contrast, the amoeboid transformation did not occur for T. vaginalis interacting with He La cells, the previously used in vitro host model cell. Initial parasitism of VECs by a single organism was followed by establishment of a monolayer of trichomonads on the host cell. Finally, parasites adhering to either VECs or HeLa cells were induced to synthesize greater amounts of the four previously described adhesins. Therefore, distinct signals after contact with either epithelial cell type leads to the morphological transformation and/or induction of adhesin synthesis by T. vaginalis.
Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichiacoli by FadR
Authors: DiRusso Concetta O. , Metzger Amy K. , Heimert Tamra L.
Published: October 2006

Fatty acid biosynthesis and fatty acid degradation in Escherichiacoliare co‐ordinately regulated at the level of transcription by the product of the fadRgene, FadR. In the present work we investigate FadR interaction with the fabA and fadL promoters. The FadR‐responsive operator within fabA, OA, was localized to a region −47 to −31 base pairs relative to the start of transcription using DNase I protection studies. The promoter and untranslated leader within fadL had two binding sites for FadR, OL1 at −25 to −9 and OL2 at −1 to +16 relative to the start of transcription. The binding affinity of FadR for OA and OL1 or OL2 was lower than that for the single site within fadB (OB) as measured using protein‐DNA gel retention assays. Overall, these experiments demonstrated that the affinity of FadR binding for DNA containing the fadB, fadL and fabA promoters was OB > OL1, OL2 > OA. We could not distinguish separate binding affinities for OL1 or OL2. We demonstrated repression of fadL transcription and activation of fabA transcription in vitro using run‐off transcription assays containing purified FadR and RNA polymerase.
Bafilomycin A1 inhibits Helicobacter pylori‐induced vacuolization of HeLa cells
Authors: Papini E. , Bugnoli M. , De Bernard M. , Figura N. , Rappuoli R. , Montecucco C.
Published: October 2006

Bafilomycin A1, a specific inhibitor of the vacuolar‐type H+‐ATPase, responsible for acidification of intra‐cellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H+‐ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.
Isolation and characterization of a topA mutant of Shigella flexneri
Authors: Bhriain Niamh Ni , Dorman Charles J.
Published: October 2006

Shigella flexneri was shown to possess a homologue of the Escherichia coli K‐12 topA gene which encodes DNA topoisomerase I. The S. flexneri topA gene was replaced by a copy of the E. coli K‐12 topA gene which has been insertionally inactivated by transposon Tn10. The topoisomer distribution of reporter plasmids showed that the presence of this topA lesion in S. flexneri correlated with an increase in the level of negative DNA supercoiling in the mutant, indicating that topoisomerase I is required to relax DNA in S. flexneri as it is in E. coli and Salmonella typhimurium. The introduction of the topA mutation also resulted in repression of transcription of a thermally regulated invasion gene located on the 230 kb virulence plasmid. In addition, the topA mutant was hypersensitive to growth medium osmolarity, was unable to grow on MacConkey indicator plates and exhibited an increased doubling time under all growth conditions tested. All of these phenotypes were fully complemented in trans by a cloned copy of the E. coli topA gene carried on a recombinant plasmid. Unlike E. coli topA mutants which acquire compensatory mutations at a high frequency, such compensatory mutations were not detected in the S. flexneri topA::Tn 10 mutant.
Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte‐like Caco‐2 and mucus‐secreting HT29 cells in culture
Authors: Eveillard Matthieu , Fourel Valérie , Bare Marie‐Claude , Kernéis Sophie , Coconnier Marie‐Hélène , Karjalainen Tuomo , Bourlioux Pierre , Servin Alain L.
Published: October 2006

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco‐2 cells and the human mucus‐secreting HT29‐MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well‐defined apical microvilli of differentiated Caco‐2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29‐MTX cells. The binding of C. difficile to Caco‐2 cells developed in parallel with the differentiation features of the Caco‐2 cells, suggesting that the protein(s) which constitute C. difficile‐binding sites are differentiation‐related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte‐like Caco‐2 cells and the human mucus‐secreting HT29‐MTX cells. A labile surface‐associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface‐associated proteins of C. difficile subjected to different culture conditions was con‐ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS‐extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface‐associated proteins in the adhe‐sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco‐2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat‐shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.
The TonB‐dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co‐evolution of receptor structure and substrate specificity
Authors: Koebnik Ralf , Hantke Klaus , Braun Volkmar
Published: October 2006

A Yersinia enterocolitica receptor mutant was isolated which is impaired in ferrichrome uptake. The receptor‐encoding gene fcuA was cloned in Escherichia coli K‐12. A fcuA mutant of Y. enterocolitica could be complemented by the cloned DNA fragment. The FcuA‐encoding region was sequenced and an open reading frame encoding 758 amino acids including a signal sequence of 36 amino acids was found. FcuA shared 34.6% amino acid sequence homology with FatA, the anguibactin receptor of Vibrio anguillarum, but only 20.6% homology with FhuA, the ferrichrome receptor of E. coli Since the structure of anguibactin differs strongly from that of ferrichrome there seems to be no co‐evolution of receptor structure and substrate specificity.

The ferrichrome receptors FcuA from Y. enterocolitica and FhuA from E. coli had slightly different substrate specificities. In contrast to FhuA from E. coli, FcuA from Y. enterocolitica was more stereoselective and failed to transport enantio ferrichrome. Three additional ferrichrome receptors were cloned from Pantoea aggiomerans (formerly Erwinia herbicola), Salmonella paratyphi B and Salmonella typhimurium. Their substrate specificity was similar but not identical.
DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics
Authors: Hatfult Graham F. , Sarkis Gary J.
Published: October 2006

Genetic studies of Mycobacterium tuberculosis and other mycobacterial pathogens have suffered from the lack of a sophisticated genetic system. To address this issue we have developed a viral system through a detailed characterization of mycobacteriophage L5, a temperate phage that infects both fast‐ and slow‐growing mycobacteria. We describe here the complete DNA sequence of the L5 genome and initial characterization of L5 virion structure and gene expression. In addition to providing a genetic‘toolbox’for the mycobacteria we find that L5 offers a new paradigm for dsDNA phages, being phenotypically temperate but employing genetic strategies for phage growth usually associated with lytic bacteriophages.
Bacterial cholesterol oxidases are able to act as flavoprotein‐linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4‐cholesten‐6‐ol‐3‐one
Authors: Molnár I. , Hayashi N. , Choi K.‐P. , Yamamoto H. , Yamashita M. , Murooka Y.
Published: October 2006

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4‐cholesten‐3‐one as a substrate and extra‐ or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA‐COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4‐cholesten‐6‐ol‐3‐one based on comparisons of its high‐performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton‐nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4‐cholesten‐6‐ol‐3‐one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6‐hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β‐hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6‐hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4‐cholesten‐3‐one and 4‐chotesten‐6‐ol‐3‐one by cholesterol oxidase, and the observed differences in the rate of formation of the 6‐hydroxy‐ketosteroid by the enzymes of different bacterial sources are also discussed.
Characterization of genes involved in molybdenum transport in Azotobacter vinelandii
Authors: Luque F. , Mitchenall L. A. , Chapman M. , Christine R. , Pau R. N.
Published: October 2006

Expression of alternative nitrogenases in Azotobacter vinelandii is repressed by molybdenum. Two strains with Tn5 insertion mutations showed alternative nitrogenase‐dependent diazotrophic growth in the presence of Mo. The mutations were in a region which contained four open reading frames (ORFs 1–4). The genetic structure and predicted products of ORFs 2, 3 and 4 are typical of the membrane‐associated elements of the ATP‐binding cassette (ABC) superfamily of transport systems. The products of 0RF3 and 0RF4 are homologous with the products of the Escherichia coli genes chlD and the partially sequenced chlJ, respectively, both of which are implicated in molybdenum transport. ORF1, which is in the relative position of bacterial permease genes commonly specifying periplasmic binding proteins, encodes a 29 kDa protein with a novel primary structure. It lacks a potential signal sequence, and its C‐terminal half consists of a tandem repeat of a segment which is homologous with the Mr 7 kDa molybdenum‐pterin binding protein Mop from Clostridium pasteurianum. This suggests that a substituted pterin may be involved in the initial capture or early metabolism of molybdenum.
Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin
Authors: Henderson Douglas P. , Payne Shelley M.
Published: October 2006

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem‐iron utilization genes allow transport of the entire haem moiety into the cell.
The two faces of Bacillus thuringiensis: insecticidal proteins and post‐exponential survival
Authors: Aronson Arthur I.
Published: October 2006

Post‐exponential Bacillus thuringiensis cells produce both an endospore and a variety of intracellular inclusions. The latter are comprised of protoxins, each being specific for the larvae of certain species from at least three orders of insects. Following ingestion of spores and inclusions, toxicity results in the spores gaining access to haemolymph, a source of nutrients suitable for germination and growth. Most B. thuringiensis subspecies contain multiple, plasmid‐encoded protoxin genes, often with several on the same plasmid. These genes have been manipulated in order to understand the basis of toxicity and specificity, information which is important to the use of these toxins as biological control agents. Some protoxin genes are in operons, and others are in close proximity, perhaps to enhance the chances of recombination, and some are on unstable plasmids. The arrangement of these genes is probably important for flexibility in the variety of protoxins packaged into inclusions by a particular subspecies and thus the capacity to adapt to changing populations of insects. Protoxins accumulate over a prolonged period during sporulation because of the sequential transcription from two promoters, each being dependent upon a specific sporulation sigma factor, the relative stability of the messenger RNA, and the synthesis of proteins which stabilize protoxins and perhaps facilitate inclusion assembly. During the post‐exponential phase, spore and inclusion formation must be balanced so as to ensure that both are available to contribute to the survival of these bacilli.