bluuebunny/crossref_metadata_2025 huggingface dataset:
The gene of the N‐acetylglucosaminidase, a Bacillus subtilis 168 cell wall hydrolase not involved in vegetative cell autolysis
Authors: Margot Philippe , Mauël Catherine , Karamata Dimitri
Published: October 2006

lytD, the structural gene of the Bacillus subtilis 168 N‐acetylglucosaminidase was localized at 310°, next to the tagABC operon. Sequence analysis revealed a monocistronic operon encoding a 95.6 kDa protein endowed with an export signal, the cleavage of which yields the monomer polypeptide (92.8 kDa) of the dimeric active form of the enzyme. Transcription is initiated at a sigma‐D (σD)‐dependent promoter and ends at a terminator common to lytD and the divergently transcribed tagABC operon. In addition, we report the sequence of the adjacent upstream ORF, transcribed in the same direction as lytD, which shows significant homology to phosphomannose isomerase‐encoding genes. Cell separation, motility, autolysis, cell wall turnover and growth were not affected in strains devoid of the N‐acetylglucosaminidase. A mutant deficient in the two most abundant autolysins, i.e. the LytC amidase and the glucosaminidase, exhibited the phenotype of the amidase‐deficient strains, revealing their non‐requirement for growth. This conclusion raises two fundamental questions: how does the cell undo the highly cross‐linked peptidoglycan so as to be able to grow, and what is the rote of the considerable amount of autolysin normally present? Possible answers to these questions are discussed.
Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis
Authors: Dybvig Kevin , Yu Hullan
Published: October 2006

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R‐M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R‐M enzymes previously found only in enteric bacteria.
Nark is a nitrite‐extrusion system involved in anaerobic nitrate respiration by Escherichia coli
Authors: Rowe John J. , Ubbink‐Kok Trees , Molenaar Douwe , Konings Wil N. , Driessen Arnold J. M.
Published: October 2006

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest‐lived radioactive isotope of nitrogen, 13N‐nitrate (half‐life = 9.96 min) and the nitrite‐sensitive fluorophore N‐(ethoxycarbonylmethyl)‐6‐methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.
Molecular basis of the optochin‐sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+‐ATPases
Authors: Fenoll Asunción , Muñoz Rosario , Garcia Ernesto , de la Campa Adela G.
Published: October 2006

The gene responsible for the optochin‐sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H+‐ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin‐sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by inter‐species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane‐bound ATPase activity of the S. pneumoniae wild‐type (OptS) strains, and found a 100‐fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.
Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba
Authors: Cázares Febe , Manning‐Cela Rebecca , Meza Isaura
Published: October 2006

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment‐length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non‐transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non‐pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA‐containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.
Control of the LexA regulon by pH: evidence for a reversible inactivation of the LexA repressor during the growth cycle of Escherichia coli
Authors: Dri Anna‐Maria , Moreau Patrice L.
Published: October 2006

The LexA repressor controls the expression of several genes, including lexA, recA, and sfiA, which are induced when exponentially growing bacteria are exposed to DNA‐damaging agents, Induction of this so‐called SOS response takes place while LexA is cleaved in a reaction that requires the RecA protein and damaged DNA. We have shown that large fluctuations in the cellular concentration of the LexA repressor and in the rate of transcription of the sfiA gene also occur spontaneously during bacterial growth in complex medium such as LB. The possibility that changes in external or internal pH may explain these fluctuations has been explored. A consistent pattern was established whereby conditions leading to either increased or decreased pH were associated with altered expression of the LexA and SfiA genes. These data can be explained by a model in which the LexA repressor exists in either of two forms in equilibrium: a form favoured at homeostatic internal pH, which has a low affinity for the operators of LexA‐controlled genes; and a form accumulated in response to a transient decrease in internal pH, which has a high affinity for operators.
Novel growth rate control of dam gene expression in Escherichia coli
Authors: Rasmussen Lene Juel , Marinus M. G. , Lobner‐Olesen Anders
Published: October 2006

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single‐copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical σ70 promoter with 18bp spacing between the ‐10 and ‐35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta‐galactosidase specific activity from a single‐copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde‐4), obtained by miniTn 10 insertion, showed the same level of β‐galactosidase activity at all growth rates tested, in contrast, growth rate‐dependent expression of the rrnB gene was unaffected by cde‐4 confirming the different modes of regulation. The cde‐4::mint Tn 10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.
The regulatory RNA gene micF is present in several species of Gram‐negative bacteria and is phylogenetically conserved
Authors: Esterling Lisa , Delihas Nicholas
Published: October 2006

micF RNA post‐transcriptionally regulates Escherichia coli outer membrane protein F (OmpF), in response to temperature increase and other environmental stress conditions, by binding to ompF mRNA and destabilizing the message. Southern analyses show that the micF gene is present in related Gram‐negative bacteria, including Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, Northern analyses Indicate that micF RNA and ompF mRNA levels are thermally regulated in several related species in a manner similar to the thermoregulation in Escherichia coli DNA sequences from Salmonella typhi, Salmonella typhimurium, and Klebsiella pneumoniae show greater than 96% homology in the micF gene when compared to the Escherichia coli micF sequence. Upstream of micF, sequences show considerable variation, although several distinct regions are highly conserved. Some of these conserved regions correspond to known binding sites for the transcription factor OmpR and the DNA‐binding protein integration host factor. In addition, E. coli micF RNA incubated with protein extracts from other species forms hetero‐logous ribonucleoproteins (RNPs). The formation of these heterologous RNPs indicates both the presence of micF RNA‐binding protein homologues in other species and a conservation of RNA‐protein recognition sites. This work demonstrates that the micF RNA regulatory system is present in other Gram‐negative bacterial species and that this system appears to be phylogenetically conserved.
Isolation and characterization of a carboxysome shell gene from Thiobacillus neapolitanus
Authors: English Robert S. , Lorbach Stanley C. , Qin Xiang , Shively Jessup M.
Published: October 2006

The gene coding for the major carboxysome shell peptide (csoS1) from Thiobacillus neapolitanus has been isolated and sequenced. Oligonucleotide primers for polymerase chain reaction (PCR) amplification of the 5’end of the gene were made possible by amino acid sequencing of the N‐terminal residues of the shell peptide. A 41 bp PCR product was used as a probe to isolate the gene. The deduced amino acid composition of the 216 bp gene shows a high degree of hydrophobicity. The gene is located within a series of three repeated regions of DNA and appears to have arisen via gene duplication. The transcript of csoS1 is approximately 400 bases in length. The shell peptide shares significant homology with Synechococcus open reading frames implicated in carboxysome structure/assembly. These open reading frames and csoS1 are related and are probably members of a carboxysome gene family.
The Lactococcus lactis sex‐factor aggregation gene cluA
Authors: Godon Jean‐Jacques , Jury Karen , Shearman Claire A. , Gasson Michael J.
Published: October 2006

A gene, cluA, was cloned from the chromosomally located sex factor of Lactococcus lactis MG1363. Sequence analysis revealed significant homology with previously described aggregation proteins in Enterococcus and Streptococcus species. The possibility that cluA was an equivalent protein involved in cell aggregation between donor and recipient bacteria during lactococcal conjugation was confirmed by its expression under the control of a heterologous promoter in L. lactis. Analysis of the homology between the CluA protein and the related proteins of Enterococcus and Streptococcus allowed a common structure for these proteins to be postulated. This consisted of five domains. Functionally conserved domains I and V act respectively as a secretary leader and C‐terminal membrane anchor. Domains II and IV are conserved at the amino acid level and probably have common structural roles whereas domain III is variable and may control binding specificity.
Two mutations that block heterocyst differentiation have different effects on akinete differentiation in Nostoc ellipsosporum
Authors: Leganés F. , Fernández‐Piñas F. , Wolk C. Peter
Published: October 2006

Evident differentiation of vegetative cells into hetero‐cysts in Anabaena sp. strain PCC 7120 is prevented by Insertions in genes hetR and hetP. Nostoc ellipsosporum possesses single copies of genes that hybridize with hetR and hetP. In mutant NE2 of N. ellipsosporum, in which hetR is interrupted by an insert, and in a double recombinant of wild‐type N. ellipsosporum with a plasmid that bears an interrupted copy of hetR, neither heterocysts nor akinetes are formed. When an intact copy of hetR from Anabaena sp. strain PCC 7120 was added to NE2 the ability to form both heterocysts and akinetes was restored, in contrast to the hetR mutant, a hetP mutant of N. ellipsosporum could form akinetes, but heterocyst formation was blocked. Use of luxAB, encoding luciferase, as a reporter, and use of luxC, luxD and luxE to generate aldehyde (a substrate for the luciferase reaction), permitted visualization of the expression of hetR at the level of single cells; hetR was expressed in akinetes.
Effects of an isogenic Zn‐metalloprotease‐deficien mutant of Legionella pneumophila in a guinea‐pig pneumonia model
Authors: Moffat Jennifer F. , Edelstein Paul H. , Regula Donald P. , Cirillo Jeffrey D. , Tompkins Lucy S.
Published: October 2006

To determine the effects, if any, of the Zn‐metallo‐protease on virulence of Legionella pneumophila infection, an isogenic mutant deficient in protease (encoded by the proA gene) was tested in an Acantha‐moeba cell model, in guinea‐pig macrophages, and in a guinea‐pig pneumonia model. The cloned proA gene was completely inactivated by insertion of a kanamycin‐resistance cassette into the protease gene of L. pneumophila AA100. This mutated gene was then introduced into the L. pneumophila chromosome by allelic exchange to form the isogenic ProA mutant AA200. AA200 showed no difference in its ability to enter, survive, or grow in Acanthamoeba and explanted guinea‐pig macrophages; neither light nor electron microscopy revealed morphological differences in the eukaryotic cells infected with the protease mutant or the wild‐type strains. The proA gene was found to be expressed in L. pneumophila during intracellular growth in amoebae by measuring the light produced from a truncated luxC gene fusion with the proA promoter. Virulence of the protease mutant was attenuated when tested in a guinea‐pig model of infection employing the intratracheal Inoculation method. AA200 was slower to cause death, grew to lower numbers in the lungs, resulted in less necrotic debris and a larger macrophage infiltrate, and was more likely to be found in association with macrophage vacuoles than the parent strain.
The ompA 5′ untranslated region impedes a major pathway for mRNA degradation in Escherichia coli
Authors: Hansen Michael J. , Chen Li‐How , Fejzo Mariena L. S. , Beiasco Joel G.
Published: October 2006

The unusual longevity of the Escherichia coli ompA transcript is determined by its 5′untranslated region (UTR), which functions In vivo as an mRNA stabilizer. Here we show that this 5′UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is Joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5′ UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome‐binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5′ UTR is not RNase III.
Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium
Authors: Perez M. Hernandez , Fomukong N. G. , Hellyer T. , Brown I. N. , Dale J. W.
Published: October 2006

A highly mobile Insertion sequence designated IS 1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20′) were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS 1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS 1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110‐derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110‐hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited In M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.
Substrate induction and catabolite repression of the Streptomyces coelicoior glycerol operon are mediated through the GyIR protein
Authors: Hindle Zoë , Smith Colin P.
Published: October 2006

The pathway for glycerol catabolism In Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol‐inducible, glucose‐repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system In which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol‐inducible, weakly glucose‐repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression In Streptomyces Is not exerted at the transcriptional level by a general ‘catabolite repressor protein’. Models for catabolite repression are discussed.
The efficiency of processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus does not require the whole prosequence, but does depend on the nature of the amino acid sequence in the region of the cleavage site
Authors: Wetmore Diana R. , Wong Sui‐Lam , Roche Rodney S.
Published: October 2006

Using deletion mutants, It is shown that part of the prosequence, the Ω‐peptide (‐4, ‐24), of the thermolysin‐like neutral protease (TNP) from Bacillus cereus, Cnp, is not required for efficient processing and secretion of fully functional mature protease. It is demonstrated that the rate and selectivity of pro‐protein processing is dependent on both the flexibility and primary sequence of the processing site. Processing is found to be particularly sensitive to the nature of the amino acid three residues upstream from the site of cleavage. A consensus sequence for TNP pro‐protein processing has been identified, which provides further Insights. Finally, a larger deletion of a portion of the Cnp prosequence upstream from the Ω‐peptide that includes amino acids conserved among TNPs reduces the rate of processing and secretion of Cnp and results in the accumulation of export‐incompetent pre‐proprotein in the cell fraction.
Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene
Authors: Zhou X. , Deng Z. , Hopwood D. A. , Kieser T.
Published: October 2006

The DNA of wild‐type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide‐host‐range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction‐modification system, because no host‐modified phages able to propagate on the wild‐type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.
Identification of a novel sugar‐H+ symport protein, FucP, for transport of L‐fucose into Escherichia coli
Authors: Gunn Frank J. , Tate Christopher G. , Henderson Peter J. F.
Published: October 2006

l‐Fucose (6‐deoxy‐l‐galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l‐fucose‐H+ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda Ol Pl promoters, induction of the T7 promoter resulted in the expression of [14C]‐l‐fucose uptake activity and the concomitant expression of a [35S]‐Met‐labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda Ol Pl promoter caused the appearance of l‐fucose‐H+ symport activity and of a Coomassie brilliant blue‐stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N‐terminal protein sequencing. The FucP protein is, therefore, a sugar‐H+ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.
Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues
Authors: Vogel Ann M. , Das Anath
Published: October 2006

Mutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1‐, VirD2‐dependent border‐nicking activity in Escherichia coli harbouring a border‐containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45–60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA‐binding domain. Deletion mutagenesis indicated that amino acids 2–16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5–27, completely inactivated VirD1.
The glucose‐starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival
Authors: Nyström Thomas
Published: October 2006

Proteins of the glucose‐starvation stimulon were identified by using two‐dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose‐starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose‐starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild‐type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose‐starvation stimulon.
Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo
Authors: Freitag Nancy E. , Portnoy Daniel A.
Published: October 2006

The PrfA transcriptional activator is an essential determinant of Listeria monocytogenes pathogenesis. prfA expression is governed by three differentially regulated promoters: prfAP1 and prfAP2, which are located immediately upstream of prfA coding sequences, and the adjacent plcA promoter via the generation of a plcA–prfA read‐through transcript. A series of promoter deletion mutants were constructed to assess the roles of prfAP1 and prfAP2. Elimination of either prfAP1 or prfAP2 resulted in altered regulation of PrfA‐regulated genes after in vitro growth. However, these mutants were fully virulent both in an animal model and in tissue culture models of infection, suggesting that the two prfA promoters are functionally redundant in vivo. In contrast, a mutant lacking both prfAP1 and prfAP2 was 100‐fold less virulent and was delayed In escape from the host vacuole. Once in the host cytoplasm, however, the double mutant was apparently normal in cell‐to‐cell spread.
A‐factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus
Authors: Horinouchi Sueharu , Beppu Teruhiko
Published: October 2006

A‐factor, containing a γ‐butyrolactone in its structure, is an autoregulatory factor or a‘microbiai hormone’controlling secondary metabolism and cellular differentiation in Streptomyces griseus. A‐factor exerts its regulatory role by binding to a specific receptor protein which, in the absence of A‐factor, acts as a repressor‐type regulator for morphological and physiological differentiation, in the signal relay leading to streptomycin production in S. griseus, the A‐factor signal is transferred from the A‐factor receptor to the upstream activation sequence of a regulatory gene, strR, in the streptomycin biosynthetic gene cluster via an A‐factor‐dependent protein that serves as a transcription factor for strR. The StrR protein thus Induced appears to activate the transcription of other streptomycin‐production genes. The presence of A‐factor homologues in a wide variety of Streptomyces species and distantly related bacteria implies the generality of γ‐butyrolactones as chemical cellular signalling molecules in microorganisms.
Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein
Authors: Hill Charles W. , Sandt Carol H. , Vlazny Donald A.
Published: October 2006

The Rhs family comprises a set of composite elements found in the chromosomes of many natural Escherichia coli strains. Five Rhs elements occur in strain K‐12. The most prominent Rhs component is a giant core open reading frame (core ORF) whose features are suggestive of a cell surface ligand‐binding protein. This hypothetical protein contains a peptide motff, xxGxxxRYxYDxxGRL(I or T)xxxx, that is repeated 28 times. A similar repeated motif is found in a Bacillus subtilis wall‐associated protein. The Rhs core ORFs consist of two distinct parts: a large N‐terminal core that is conserved in all Rhs elements, and a smaller C‐terminus that Is highly variable. Distinctive G+C contents of Rhs components indicate that the elements have a recent origin outside the E. coli species, and that they are composites assembled from segments with very different evolutionary histories. The Rhs cores fail into three sub‐families that are mutually more than 20% divergent Downstream of the core ORF is a second, much shorter ORF. Like the adjacent core extension, these are highly variable. In most examples, the hypothetical product of this ORF has a candidate signal sequence for transport across the cytoplasmic membrane. Another Rhs component, the 1.3 kb H‐rpt, has features typical of insertion sequences. Structures homologous to H‐rpt have been detected in other bacterial genera, such as Vibrio and Salmonella, where they are associated with loci that determine O‐antigen variation.
Unique fimbriae‐like structures encoded by sefD of the SEF14 fimbrial gene cluster of Salmonella enteritidis
Authors: Clouthler S. C. , Collinson S. K. , Kay W. W.
Published: October 2006

The SEF14 gene cluster of Salmonella enteritidis was recently shown to contain three genes, sefABC, encoding a unique fimbrin, and proteins homologous to fimbrial chaperones and outer membrane proteins (ushers), respectively. A fourth open reading frame, designated sefD, was found immediately downstream of sefABC and overlapping sefC. The translated protein sequence of sefD was unique, but the composition was similar to that of other bacterial fimbriae. SefD was produced in abundance by wild‐type S. enteritidis as shown by Western blot analysis using antibodies raised to affinity‐purified, recombinant SefD. Furthermore, unusually long, thin, fimbriae‐like structures were evident on S. enteritidis and Escherichia coli by immunoelectron microscopy, but in other bacterial species SefD was expressed as amorphous material. Therefore, in S. enteritidis and E. coli, SefD is the predominant structural subunit of SEF18. The SEF18 fimbriae‐like structures were shown to be serologically distinct from the three known S. enteritidis fimbriae SEF14, SEF17 and SEF21. Furthermore, SEF18 was still produced in set A insertion mutants, indicating that SEF14 and SEF18 were structurally distinct. Thus, the SEF14 gene cluster is the first example in the Enterobacteriaceae of a gene cluster that encodes two fimbrin‐like proteins, which are assembled into two distinct cell‐surface structures, SEF14 and SEF1B. DNA hybridization and Western blot analyses showed that SefD was widely distributed among the Enterobacteriaceae and was present in E. coli, Shigella, Enterobacter, Citrobacter, Erwinia, Hafnia, Klebsiella, Providencia, and Proteus but not in the non‐Enterobacteriaceae Gram‐negative bacteria Pseudomonas and Aeromonas, or in Gram‐positive bacteria Bacillus or Staphylococcus. Immunoelectron microscopy revealed that sefD was also present on the surface of Providencia and Klebsiella but did not appear filamentous. This is the first instance of highly conserved, thin fimbriaelike structers which are ubiquitous among the Enterobacteriaceae.
The functional units of a peptostreptococcal protein L
Authors: Murphy Jonathan P. , Duggleby Clive J. , Atkinson Max A. , Trowern Angus R. , Atkinson Tony , Goward Christopher R.
Published: October 2006

Protein L is a cell‐surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin‐binding units have high amino acid sequence identity with the albumin‐binding units of streptococcal cell‐surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.
A Reb1p‐binding site is required for efficient activation of the yeast RAP1 gene, but multiple binding sites for Rap1p are not essential
Authors: Graham Ian R. , Chambers Alistair
Published: October 2006

The Saccharomyces cersvislae RAP1 protein (Rap1p) is a key multifunctional transcription factor. Using gel retardation analysis, four binding sites for RAP1p have been identified within the promoter of the RAP1 gene. These sites are located downstream of a binding site for the transcription factor Reb1p. The Reb1p site and an associated AT‐rich region are important for transcriptional activation, but deletion of three of the RAP1 p‐binding sites had little effect on promoter activity. The activity of the RAP1 promoter has been analysed in a yeast strain (YDS410) that contains a temperature‐sensitive mutation In the RAP1 gene. This mutation renders the DNA‐binding activity of Rapip temperature dependent. When YDS410 was grown at a semi‐permissive temperature (30°C), the activity of the RAP1 promoter increased by approximately 170%, compared with the same strain grown at the permissive temperature (25°C). A RAP1 promoter in which three of the four RAP1 p‐binding sites had been deleted, showed only a small increase in activity in the same experiment. These data confirm that Rap1p is not required for activation of the RAP1 gene, and suggest a role for Rap1p In negative auto‐regulation.
Genetic and molecular characterization of capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 3
Authors: Dillard Joseph P. , Yother Janet
Published: October 2006

To achieve a better understanding of the genetics of capsular polysaccharide synthesis in Streptococcus pneumoniae, we have identified and characterized mutants deficient in type 3 capsule production. We Identified a clone that restored encapsulation in one of our mutants and in a mutant deficient in UDP‐glucose dehydrogenase. By hybridization, we developed a chromosomal map of the type 3‐specific region and identified a flanking region containing DNA common to all capsule types examined. Insertion mutations were used to identify chromosomal loci required for capsule synthesis, and to map transcription within the region. Using non‐destructive insertions linked to type‐specific genes of type 2, 3, or 5, we were able to select for the transformation of all necessary genes specific for capsule type. Our data provide molecular evidence to show that all the type‐specific genes are linked in a cassette and can be transferred as a unit during transformation.
Bacterial binding protein‐dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins
Authors: Saurin William , Köster Wolfgang , Dassai Elle
Published: October 2006

Bacterial binding protein‐dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA … G ………‐I ‐ LP, located approximately 100 residues from the C‐terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic α‐helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures.
Light‐regulated promoters from Synechocystis PCC6803 share a consensus motif involved in photoregulation
Authors: Marraccini Pierre , Cassier‐Chauvat Corinne , Bulteau Stéphane , Chavez Sebastian , Chauvat Franck
Published: October 2006

A library of Synechocystis PCC6803 (S.6803) DNA cioned in front of the promoterless cat reporter gene of the plasmid pFF11 was used to transform S.6803 to high light‐dependent resistance to chloramphenicol. In five clones harbouring a stably replicating pFF11‐derived plasmid, this phenotype occurred independently of the photosystem II electron transport and resulted from the correlated increase of CAT activity level and cat mRNA accumulation. The five promoter inserts contained no Escherichia collω70 promoter element, in agreement with their lack of activity in this organism, but shared two conserved motifs. Two secondary mutations, which restored light‐regulated promoter activity to an inactive mutant of the smallest insert, mapped within one of the common motifs, emphasizing the probable involvement of this element in photoregulation.
A topological model for the high‐affinity nickel transporter of Alcaligenes eutrophus
Authors: Eitinger Thomas , Friedrich Bärbel
Published: October 2006

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy‐dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino‐terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β‐galactosidase (LacZ). The enzymatic activity of 16 HoxN‐PhoA and 15 HoxN‐LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two‐dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.
Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae
Authors: Higgins Dairen E. , DiRita Victor J.
Published: October 2006

Co‐ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR‐regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR‐dependent toxT expression are discussed.
Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli
Authors: Bénard L. , Philippe C. , Dondon L. , Grunberg‐Manago M. , Ehresmann B. , Ehresmann C. , Portier C.
Published: October 2006

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome‐binding site. By measuring the effect of mutations on the expression of a translational rpsO‐lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.
Common features of the NAD‐binding and catalytic site of ADP‐ribosylating toxins
Authors: Domenighini Mario , Magagnoli Claudia , Pizza Mariagrazia , Rappuoli Rino
Published: October 2006

Computer analysis of the three‐dimensional structure of ADP‐ribosylating toxins showed that in all toxins the NAD‐binding site is located in a cavity. This cavity consists of 16 contiguous amino acids that form an a‐helix bent over β‐strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD‐binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophillc residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat‐labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP‐ribosylation evolved to work on different target proteins.
Probing the structure‐activity relationship of Escherichia coli LT‐A by site‐directed mutagenesis
Authors: Pizza Mariagrazia , Domenighini Mario , Hol Wim , Giannelli Valentine , Fontana Maria Rita , Giuliani Marzia M. , Magagnoli Claudia , Peppoloni Samuele , Manetti Roberto , Rappuoli Rino
Published: October 2006

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat‐labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site‐directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non‐toxic new molecules containing mutations of single amino acids such as Val‐53 → Glu or Asp, Ser‐63 → Lys, Val‐97 → Lys, Tyr‐104 → Lys or Asp, and Ser‐14 → Lys or Glu. This group also included mutations in amino acids such as Arg‐7, Glu‐110 and Glu‐112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu‐41 → Phe, Ala‐45 → Tyr or Glu, Val‐53 → Tyr, Val‐60 → Gly, Ser‐68 → Pro, His‐70 → Pro, Val‐97 → Tyr and Ser‐114 → Tyr. The third group contained those molecules that maintained a wild‐type level of toxicity in spite of the mutations introduced: Arg‐54 → Lys or Ala, Tyr‐59 → Met, Ser‐68 → Lys, Ala‐72 → Arg, His or Asp and Arg‐192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non‐toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.
New clusters of genes required for gliding motility in Myxococcus xanthus
Authors: MacNeil Spencer D. , Calara Federico , Hartzell Patricia L.
Published: October 2006

Gilding is the directed movement of cells across surfaces which occurs in the absence of external organelles such as flagella. Gliding of the complex prokaryote, Myxococcus xanthus, results from the action of two independent sets of genes known as the A (adventurous motility) and S (social motility) genes. Strains with mutations in both systems (A−S−) do not spread on agar surfaces because both individual and group movement is abolished. To generate regulated, transcriptional fusions with operons including A and S genes, we introduced TN5‐lac into A− and S− strains to obtain non‐motile A−S::Tn5‐lac and A::Tn5‐lac S− double mutants. These insertions identify five separate clusters of A genes and three separate clusters of S genes on the M. xanthus genome. Some Tn5‐lac insertions map near two of the five previously identified motility gene clusters, but at least five new clusters were identified in this search. Single mutations at only one locus, mgIA, block motility; the mgIA locus is epistatic to A and S motility genes. A‐and S‐Tn5‐lac insertions were transduced into mgl+ and δmgl strains. The levels of β‐galactosidase activity produced from each A‐or S‐Tn5‐lac insertion are similar in otherwise isogenic mgl+ and δmgl strains, showing that MgIA does not regulate the transcription of many A and S genes.
The FeSII protein of Azotobacter vinelandii is not essential for aerobic nitrogen fixation, but confers significant protection to oxygen‐mediated inactivation of nitrogenase in vitro and in vivo
Authors: Moshiri Farhad , Kim John W. , Fu Changlin , Maier Robert J.
Published: October 2006

Summary The FeSII protein of Azotobacter vinelandii has been proposed to mediate the ‘conformational protection’ of the molybdenum‐dependent nitrogenase components against oxygen inactivation. We have cloned and characterized the structural gene for the FeSII protein (the fesII Iocus). Hybridization studies did not reveal the presence of fesII‐like genes in a number of diverse species of well‐studied nitrogen‐fixing bacteria, with the exception of Azotobacter chroococcum. The fesll locus is transcriptionally expressed during both nitrogen fixing and non‐nitrogen fixing conditions, although the level of its message is up‐regulated by approximately 2.5‐fold during nitrogen fixation. The promoter region was identified by primer extension analysis, and is similar to other σ70‐type promoters. Mutants devoid of the FeSII protein were constructed. These mutants possessed growth characteristics on a variety of carbon substrates during non‐diazotrophic as well as diazotrophic growth that were essentially indistinguishable from the wild‐type strain. Nevertheless, the nitrogenase activity in cell‐free extracts is significantly more sensitive to irreversible oxygen inactivation in the mutants as compared with the wild type. When treated with 250 mM NaCI (a condition known to dissociate FeSII from nitrogenase components), the wild‐type and mutant extracts were equally hypersensitive to oxygen Inactivation. Upon energy starvation, conditions in which ‘respiratory protection’ is inoperable, the MoFe and Fe proteins of nitrogenase are degraded much more rapidly in vivo in the deletion mutants, compared to the wild type. Strains relying on either the vanadium or the ‘iron‐only’ alternative nitrogenases exhibited similar growth rates irrespective of the presence of absence of the FeSII protein, and the in vitro inactivation of the vanadium nitrogenase components was not affected by the lack of the FeSII protein. All in all, these results are consistent with a model whereby ‘respiratory protection’ is the major physiological mechanism responsible for the protection of all three nitrogenases during energy supplemented growth. Upon energy starvation, however, ‘conformational protection’ mediated by the FeSII protein is capable of temporarily protecting the conventional molybdenum nitrogenase components from inactivation and subsequent degradation.