bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010
Authors: Recchia Gavin D. , Hall Ruth M.
Published: October 2006

Gene cassettes are mobile DNA elements which contain a specific recombination site, a 59‐base element, recognized by the site‐specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette‐associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (int), and it is likely that the cassette was inserted via a single Int‐mediated recombination event between the 59‐base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette‐associated recombination site was inactivated by the insertion, and Int‐mediated excision of the aadB cassette from this non‐specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.
Virulence studies on chromosomal α‐toxin and Θ‐toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α‐toxin in Clostridium perfringens‐mediated gas gangrene
Authors: Awad Miilena M. , Bryant Amy E. , Stevens Dennis L. , Rood Julian I.
Published: October 2006

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin‐resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen‐labile haemolysin known as Θ‐toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α‐toxin or phospholipase C. The resultant mutants failed to produce detectable Θ‐toxin or α‐toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild‐type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α‐toxin in gas gangrene or clostridial myonecrosis.
Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis
Authors: van Sinderen Douwe , Kiewiet Rense , Venema Gerard
Published: October 2006

Despite the lack of involvement of the competence‐specific, membrane‐associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein, nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream of nucA. The expression of nucB, specifying an extracellular DNase, which was cloned on the basis of its homology to nucA, was shown to be sporulation‐specific and dependent on the gene products of spo0A and spollG, the latter constituting an operon responsible for the synthesis of the mother‐cell‐specific sigma factor σE. The observed differential expression of nucA and nucB demarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post‐exponential growth‐phase processes.
A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae
Authors: Alagramam Kumar , Naider Fred , Becker Jeffrey M.
Published: October 2006

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N‐end‐rule pathway of the ubiquitin‐dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N‐end‐rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N‐end‐rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.
The pho regulon of Shigella flexneri
Authors: Scholten Monica , Janssen Riny , Bogaarts Corné , van Strien Joost , Tommassen Jan
Published: October 2006

Growth of Escherichia coli K‐12 in low‐phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (Pi). This response is controlled by a two‐component regulatory system of which PhoB and PhoR are the response‐regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for Pi, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site‐directed mutagenesis revealed that one of these deviations, i.e. Leu‐172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.
Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains
Authors: Pannekoek Yvonne , Dankert Jacob , van Putten Jos P. M.
Published: October 2006

Here we report the cloning and expression, in Escherichia coli, of PCR‐amplified DNA encoding the 63‐kDa stress‐inducible protein of Neisseria gonorrhoeae strains VP1 and PiD2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541‐544 amino acids corresponding to a protein of approximately 57 000 Da. The various neisserial proteins were >98% identical at the amino acid level and showed extensive homology with proteins belonging to the HspSO heat‐shock‐protein family. We constructed defined glutathione S‐transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria‐unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients’sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.
Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product n required for assembly of all c‐type cytochromes, including cytochrome c1
Authors: Page M. Dudley , Ferguson Stuart J.
Published: October 2006

A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c‐type cytochromes and accumulated periplasmic apocytochrome C550, but the membrane‐bound holocytochromes c1 and C552 were present at 5‐10% of the levels observed in wild‐type ceils, DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild‐type DNA library. A hybridizing 3.05 kb Bam HI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DPI08 and the B. japonicum cycH mutant C0X3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.
Sequence analysis of subunits of the membrane‐bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron‐carrying arm of a redox loop
Authors: Berks Ben C. , Page M. Dudley , Richardson David J. , Reilly Ann , Cavill Alan , Outen Fenno , Ferguson Stuart J.
Published: October 2006

Three genes, narH, narJ and narl of the membrane‐bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (β, putative δ and γ) subunits of the two membrane‐bound nitrate reductases of the enteric bacterium Escherichia coli. AU iron‐sulphur cluster ligands proposed for the E. coliβ subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly α‐helical structure. Comparison of T. pantotropha Narl wilh the b‐haembinding integral membrane subunits of the E. coli enzymes allows assignment of His‐53, His‐63, His‐186 and His‐204 (T. pantotropha Narl numbering) as b‐haem axial ligands and the construction of a three‐dimensional model of this subunit. This model, in which the two b‐haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane‐bound uptake hydrogenases and membrane‐bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate‐reducing bacteria has structural resemblance to Narl.
Coevolution of RNA helix stability and Shine‐Dalgarno complementarity in a translational start region
Authors: Olsthoorn René C. L. , Zoog Stephen , van Duin Jan
Published: October 2006

The initiation legion of the coat‐protein gene of RNA bacteriophage MS2 adopts a well‐defined hairpin structure with the start codon occupying the loop position, while the Shine‐Dalgarno (SD) sequence is part of the stem. In a previous study, we introduced mutations in this hairpin that changed its thermo dynamic stability. The resulting phages evolved to regain the wild‐type stability by second‐site compensatory substitutions. Neither the original nor the suppressor mutations were in the SD region. In the present analysis, we have made changes in the SD region that shorten or extend its complementarity to the 3 end of 16S rRNA and monitored their evolution to a stable pseudorevertant species. Phages in which the SD complementarity was decreased evolved an initiator hairpin of lower stability than wild type while those in which the complementarity was extended evolved a hairpin with an increased stability. We conclude that weaker SD sequences still allow maximal translation if the secondary structure of the ribosome‐landing site is destabilized accordingly. Alternatively, translation‐initiation regions with a stronger secondary structure still allow maximal expression, if the SD complementarity is extended. These findings support a previously published model in which the SD Interaction helps the ribosome to melt the structure in a translation‐initiation region.
Characterization of RNA polymerse and two sigma‐factor genes from Mycobacterium smegmatis
Authors: Predich Mima , Doukhan Laurence , Nair Gopalan , Smith Issar
Published: October 2006

A search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major Sigma factors in diverse prokaryotes has identified two such determinants. Both genes are expressed in exponentially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these Sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major Sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly active B. subtilis RNA polymerase holoenzyme was unable to transcribe this gene.
Structure‐function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression
Authors: Hufton Simon E. , Ward Richard J. , Bunce Nigel A. C. , Armstrong John T. , Fletcher Anthony J. P. , Glass Robert E.
Published: October 2006

We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5‐like bacteriophage BF23, and vitamin B12 (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non‐functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz, Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two‐dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic β‐strand analysis and sequence conservation amongst the TonB‐dependent receptors). In addition, we report that the extreme C‐terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane‐spanning β‐sheet with residue L588 situated on its hydrophobic surface. Two of the C‐terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.
Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram‐positive bacteria?
Authors: Hueck Christoph J. , Hillen Wolfgang
Published: October 2006

Three components involved in catabolite repression (CR) of gene expression in Bacillus have been identified. The cis‐acting catabolite responsive element (CRE), which is present in many genes encoding carbon catabolic enzymes in various species of the Gram‐positive bacteria, mediates CR of several genes in Bacillus subtilis, Bacillus megaterium, and Staphylococcus xylosus. CR of most genes regulated via CRE is also affected by the trans‐acting factors CcpA and HPr. Similarities between CcpA and Lac and Gal repressors suggest binding of CcpA to CRE. HPr, a component of the phosphoenol pyruvate:sugar phosphotransferase system, undergoes regulatory phosphorylation at a serine residue by a fcuctose‐1,6‐diphosphate‐activated kinase. A mutant of HPr, which is not phosphorylatable at this position because of an exchange of serine to alanine, lacks CR of several catabolic activities. This mutant phenotype is similar to the one exhibited by a ccpA mutant. Direct protein‐protein interaction between CcpA and HPr(Ser‐P) was recently demonstrated and constitutes a link between metabolic activity and CR.
Alkaliphiles:‘basic’molecular problems of pH tolerance and bioenergetics
Authors: Krulwich Terry Ann
Published: October 2006

Alkaliphilic Bacillus species provide experimental opportunities for examination of physiological processes under conditions in which the stress of the extreme environment brings issues of general biological importance into special focus. The alkaliphile, like many other cells, uses Na+/H+ antiporters in pH regulation, but its array of these porters, and other ion‐flux pathways that energize and support their activity, result in an extraordinary capacity for pH homeostasis; this process nonetheless becomes the factor that limits growth at the upper edge of the pH range. Above pH 9.5, aerobic alkaliphiles maintain a cytoplasmic pH that is two or more units below the external pH. This chemiosmotically adverse δpH is bypassed by use of an electrochemical gradient of Na+ rather than of protons to energize solute uptake and motility. By contrast, ATP synthesis occurs via completely proton‐coupled oxidative phosphorylation that proceeds just as well, or better, at pH10 and above as it does in the same bacteria growing at lower pH, without the adverse pH gradient. Various mechanisms that might explain this conundrum are described, and the current state of the evidence supporting them is summarized.
Regulation of gene expression by trans‐encoded antisense RNAs
Authors: Delihas Nicholas
Published: October 2006

Members of a class of antisense RNAs are encoded by genes that are located at loci other than those of their target genes. Three examples of antisense RNA genes are discussed here. micF is found in Escherichia coli and other bacteria and functions to control outer membrane protein F levels in response to environmental stimuli. dicF is also found in E. coli and is involved in the regulation of cell division, lin‐4 is found in the nematode Caenorhabditis elegans and functions during larval development. Nucleotide sequences of at least two of these genes appear to be phylogenetically conserved. The trans‐encoded antisense RNAs are small RNAs which display only partial complementarity to their target RNAs. Models for RNA/RNA interactions have been proposed. It is possible that currently known unlinked antisense RNA genes are part of a larger class of heretofore undiscovered regulatory RNA genes. Possible ways of detecting other unlinked antisense RNA genes are discussed.
Identification of a novel cellulose‐binding domain the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima
Authors: Winterhalter Christoph , Heinrich Peter , Candussio Anton , Wich Günther , Liebl Wolfgang
Published: October 2006

A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo‐1,4‐β‐D‐xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined. With a half‐life of about 40 min at 90°C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059‐amino‐acid (˜120 kDa) modular enzyme composed of an N‐terminal signal peptide and five domains, in the order A1‐A2‐B‐C1‐C2. By comparison with other xylanases of family 10 of glycosyl hydrolases, the central ˜340‐amino‐acid part (domain B) of XynA represents the catalytic domain. The N terminal ˜150‐amino‐acid repeated domains (A1‐A2) have no significant similarity to the C‐terminal ˜170‐amino‐acid repeated domains (C1‐C2). Cellulose‐binding studies with truncated XynA derivatives and hybrid proteins indicated that the C‐terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose‐binding domain (CBD) and thus are CBDS of a novel type. Structurally related protein segments which are probably also CBDs were found in other multi‐domain xylanolytic enzymes. Deletion of the N‐terminal repeated domains or of all the non‐catalytic domains resulted In substantially reduced tbermostability while a truncated xylanase derivative lacking the C‐terminal tandem repeat was as thermostable as the full‐length enzyme. It is argued that the multidomain organization of some enzymes may be one of the strategies adopted by thermophiles to protect their proteins against thermal denaturation.
Induction of phospholipase‐ and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD
Authors: Givskov Michael , Eberl Leo , Christiansen Gunna , Benedik Michael J. , Molin Søren
Published: October 2006

When a liquid culture of Serratia spp. reaches the last part of the logarithmic phase of growth it induces the synthesis of several extracellular hydrolytic enzymes. In this communication we show that synthesis and secretion of the extracellular phospholipase is coupled to expression of flagella. Expression of flagella is demonstrated to follow a growth‐phase‐dependent pattern. Cloning, complementation studies and DNA‐sequencing analysis has identified a genetic region in Serratia liquefaciens which exhibits extensive homology to the Escherichia coli flhD flagellar master operon. Interruption of the chromosomal flhD operon in S. liquefaciens results in non‐flagellated and phospholipase‐negative cells, but the synthesis of other exoenzymes is not affected. By placing the flhD operon under the control of a foreign inducible promoter we have shown that increased transcription through the flhD operon leads to induction of flagellar synthesis and phospholipase expression.
Plasmodium falciparum protein associated with the invasion junction contains a conserved oxidoreductase domain
Authors: Hudson‐Taylor Diana E. , Dolan Stephen A. , Klotz Francis W. , Fujioka Hisashi , Aikawa Masamichi , Koonin Eugene V. , Miller Louis H.
Published: October 2006

The merozoite cap protein‐1 (MCP‐1) of Plasmodium falciparum follows the distribution of the moving Junction during invasion of erythrocytes. We have cloned the gene encoding this protein from a cDNA library using a monoclonal antibody. The protein lacks a signal sequence and has no predicted trans‐membrane domains; none of the antisera reacts with the surfaces of intact merozoites, indicating that the cap distribution is submembranous. MCP‐1 is divided into three domains. The N‐terminal domain includes a 52‐amino‐acid region that is highly conserved in a large family of bacterial and eukaryotic proteins. Based on the known functions of two proteins of this family and the pattern of amino acid conservation, it is predicted that this domain may possess oxido‐reductase activity, since the active cysteine residue of this domain is invariant in all proteins of the family. The other two domains of MCP‐1 are not found in any other members of this protein family and may reflect the specific function of MCP‐1 in invasion. The middle domain is negatively charged and enriched in glutamate; the C‐terminal domain is positively charged and enriched in lysine. By virtue of its positive charge, the C‐terminal domain resembles domains in some cytoskeleton‐associated proteins and may mediate the interaction of MCP‐1 with cytoskeleton in Plasmodium.
Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF‐ defect
Authors: Kashefi Kazem , Hartzell Patricia L.
Published: October 2006

An insertion of transposon Tn5‐lac, ω4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development. Sequence analysis of the junction of ω4519 with M. xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium. When frzF‐ (or frzCD‐) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild‐type complement of spores. At higher densities, frzF::ω519 cells form‘frizzy’aggregates and produce 80–90% of the wild‐type complement of spores. In contrast, when cells with both a frzF‐ (or frzCD‐) and an sgtA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild‐type complement of heat‐resistant spores. In addition to suppressing the density dependence of fruiting‐body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF‐ mutations and a frzCD‐ mutation. In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz‐ mutations. Thus, the suppression of frz‐ mutations by sgl‐ mutations is allele‐specific, and depends on the sgl allele, but not the frz allele. Because the phenotypes of frz‐ mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.
The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence
Authors: Stojijkovic Igor , Hwa Vivian , de Saint Martin Luc , O'Gaora Peadar , Nassif Xavier , Heffron Fred , So Magdalene
Published: October 2006

The Neisseris meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin‐requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB‐dependent receptors of Gram‐negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB‐dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin‐supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.
ComC is required for the processing and translocation of ComGC, a pilin‐like competence protein of Bacillus subtilis
Authors: Chung Y. S. , Dubnau D.
Published: October 2006

ComGC is a cell surface‐localized protein required for DNA binding during transformation in Bacillus subtilis. It resembles type IV prepilins in its N‐terminal domain, particularly in the amino acid sequence surrounding the processing cleavage sites of these proteins. ComC is another protein required for DNA binding, which resembles the processing proteases that cleave type IV prepilins. We show here that ComGC is processed in competent cells and that this processing requires ComC. We also demonstrate that the PilD protein of Neisseria gonorrhoeae, a ComC homologue, can process ComGC in Escherichia coli, and that the ComC protein itself is the only B. subtilis protein needed to accomplish cleavage of ComGC in the latter organism. Based on NaOH‐solubility studies, we have shown that in the absence of ComC, but in the presence of all other competence proteins, B. subtilis is incapable of correctly translocating ComGC to the outer face of the cell membrane. Finally, we show that ComGC can be cross‐linked to yield a form with higher molecular mass, possibly a dimer, and present evidence suggesting that formation of the higher mass complex takes place in the membrane, prior to translocation. Formation of this complex does not require ComC or any of the comG products, other than ComGC itself.
AmpD, essential for both β‐lactamase regulation and cell wall recycling, is a novel cytosolic N‐acetylmuramyl‐L‐alanine amidase
Authors: Jacobs Christine , Joris Bernard , Jamin Marc , Klarsov Klaus , van Beeumen Jozef , Mengin‐Lecreulx Dominique , van Heijenoort Jean , Park James T. , Normark Staffan , Frère Jean‐Marie
Published: October 2006

In enterobacteria, the ampD gene encodes a cytosolic protein which acts as a negative regulator of β‐lactamase expression. It is shown here that the AmpD protein is a novel N‐acetylmuramyl‐L‐alanine amidase (E.C.3.5.1.28) participating in the intracellular recycling of peptido‐glycan fragments. Surprisingly, AmpD exhibits an exclusive specificity for substrates containing anhydro muramic acid. This anhydro bond is mainly found in the peptidoglycan degradation products formed by the periplasmic lytic transglycosylases and thus might behave as a‘recycling tag’allowing the enzyme to distinguish these fragments from the newly synthesized peptidoglycan precursors. The AmpD substrate (or substrates) which accumulates in the absence of the corresponding enzymatic activity acts as an intracellular positive effector for β‐lactamase expression and might represent an element of a communication network between the chromosome and the cell wall peptidoglycan.
Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901
Authors: Möhrle V. , Roos U. , Bormann C.
Published: October 2006

Expression of genes involved in nikkomycin production in Streptomyces tendae was investigated by two‐dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when nikkomycin was produced; these proteins were not detected in non‐producing mutants. N‐terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two‐dimensional gels. Protein P8 was identified as l‐histidine amino‐transferase (HisAT), which has been previously correlated with nikkomycin production. By using oligo‐nucleotide probes deduced from the N‐terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tü901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene‐disrupted transformants did not produce nikkomycin, indicating that proteins P1 and P2 are essential for nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in nikkomycin production.
Identification of phosphoproteins in Escherichia coli
Authors: Freestone P. , Grant S. , Toth I. , Norris V.
Published: October 2006

The substrates of ion‐ and lipld‐stimulated protein kinase activity in extracts of Escherichia coli were purified by chromatography. Subsequent N‐terminal sequencing suggests that these substrates include the following: a novel 80kDa protein co‐purifying with RNA polymerase but partially homologous to elongation factor G; a protein with an apparent molecular weight of 65kDa identified as the ribosomal protein S1; and a 32 kDa protein identified as succinyl CoA synthetase, a key enzyme in the tricarboxylic acid cycle. The phosphorylation of these three proteins was markedly stimulated by the addition of manganese, and occurred on threonine, serine or tyrosine residues as indicated by the stability of the phosphoresidues during acid treatment. In addition, a calcium‐stimulated protein of 70kDa was identified as the heat‐shook protein DnaK, and a 17kDa lipid‐stimulated phosphoprotein as nucleotide diphosphate kinase.
Group B streptococci adhere to a variant of fibronectin attached to a solid phase
Authors: Tamura Glen S. , Rubens Craig E.
Published: October 2006

Group B streptococci (GBS) are the leading cause of neonatal pneumonia and meningitis. Adherence of GBS to host tissues may play an important role in the pathogenesis of infection. The host molecules which mediate GBS adherence to host tissues are unknown. Many bacterial pathogens adhere to fibronectin, an important component of the extracellular matrix (ECM). Some pathogens adhere to both immobilized and soluble fibronectin, while others adhere to immobilized fibronectin, but not to soluble fibronectin. Previous data indicated that GBS do not adhere to soluble fibronectin. We studied the ability of GBS to adhere to immobilized fibronectin. Forty‐five per cent of the input inoculum of COH1, a virulent GBS isolate, adhered to fibronectin immobilized on polystyrene. COH1 did not adhere to the other ECM proteins tested (laminin, type I collagen, vitronectin, and tenascin). Nine out of nine GBS strains from human sources tested adhered specifically to fibronectin at levels varying from 4–60%. We considered the possibility that GBS were adherent to a contaminant in the fibronectin preparation. Properties of fibronectin, including the presence of an immunologic epitope of fibronectin and binding to collagen, were verified to be properties of the molecule to which GBS adhere. COH1 adhered to fibronectin captured by a monoclonal antibody to fibronectin (FN‐15), confirming that the molecule to which GBS adhere bears immunologic determinants of fibronectin. Adherence of COH1 to fibronectin was inhibited by collagen, confirming that the molecule to which GBS adhere binds to collagen. These data strongly suggest that GBS adhere to fibronectin, and not to a contaminant. Protein blot analysis revealed that GBS were adherent to a high‐molecular‐weight variant of non‐reduced fibronectin monomers and dimers. GBS did not adhere to reduced fibronectin monomers. We conclude that GBS adhere to a variant of plasma fibronectin when attached to a solid phase.
Analysis of a chemotaxis operon in Rhizobium meliloti*
Authors: Greek Michaela , Platzer Josef , Sourjik Victor , Schmitt Rüdiger
Published: October 2006

Genes controlling chemotaxis towards L‐amino acids and d‐mannitol in Rhizobium meliloti have been identified by Tn5 insertions that lead to chemotaxis‐deficient mutants. The tagged genes span an 8.7 kbp region that has been sequenced. These genes are part of a large operon containing three novel open reading frames, orf1, orf2 and orf9, and six familiar chemotaxis (che) genes, cheY1‐cheA‐cheW‐cheR‐cheB‐cheY2, that have been assigned by their similarity to known Escherichia coli genes. The second copy of cheY may be part of a second signalling chain; orf1 and orf2 encode sequence motifs that resemble the signalling domain of E. coli MCPs (methyl‐accepting chemotaxis proteins), while the product of orf9 may contain a transmembrane domain. No protein methylation has been observed in Rhizobium meliloti in response to l‐amino acids. However, the presence of cheR (methyltransferase gene) and cheB (methyl‐esterase gene) suggested that MCPs are likely components of the chemotactic response in R. meliloti. Therefore, it is postulated that two chemotaxis pathways are functional in R meliloti: one responds to l‐amino acids via ORF1‐ORF2, whereas the other (probably responding to specific plant exudates) acts via MCP‐like receptors, and both interact with the central components CheW‐CheA‐CheY1 and/or CheY2.
Interspecies recombination In nature: a meningococcus that has acquired a gonococcal PIB porin
Authors: Vázquez Julio A‐ , Berron Sonsoles , O'Rourke Maria , Carpenter Gill , Feil Edward , Smith Noel H. , Spratt Brian G.
Published: October 2006

A. vaginal isolate of Neisseria has been reported to resemble Neisseria meningitidis in biochemical characteristics but to react with serological reagents that are specific to the PI porin from Neisseria gonorrhoeae. We have confirmed that this isolate has the biochemical attributes of a meningococcus and have shown that it clusters among meningococcal Isolates on a dendrogram based on isoenzyme variation within housekeeping enzymes from populations of N. meningitidis and N. gonorrhoeae. Furthermore, the sequences of the fbp and adk genes were typical of those of N. meningitidis and were distinct from those of N. gonorrhoeae. However, the porB gene was very similar to the por genes of N. gonorrhoeae isolates that express the PIB class of outer‐membrane porin (differing from one gonococcal por allele at only a single nucleotide site), and was clearly distinct from the porB genes of N. meningitidis. The isolate therefore appears to be a typical meningococcus, except that its porB gene has been replaced with the por gene from a gonococcus.
Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis
Authors: Wilson Theresa M. , de Lisle Geoffrey W. , Collins Desmond M.
Published: October 2006

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea‐pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH‐resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH‐sensitive strain was virulent for guinea‐pigs, the INH‐resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.
Molecular population genetic analysis of the enn subdivision of group A streptococcal emm‐like genes: horizontal gene transfer and restricted variation among enn genes
Authors: Whatmore Adrian M. , Kapur Vivek , Musser James M. , Kehoe Michael A.
Published: October 2006

The group A streptococcal emm‐like genes, which encode the cell‐surface M and M‐like proteins, are divided into distinct mrp, emm and enn subdivisions and are clustered together in a region of the chromosome called the vir regulon. In order to understand the mechanisms involved in the evolution of emm‐like genes, a 180bp fragment of the 5 variable region of the enn gene was characterized in 31 strains for which emm sequences and multilocus enzyme electrophoretic profiles have been previously determined. The results demonstrate that nucleotide polymorphisms at the enn locus are generated predominantly by point mutations and short deletions or insertions, and that variation among enn and emm genes has arisen by similar mechanisms. However, diversity at the enn locus is restricted in comparison to the emm locus. Moreover, there is strong evidence for intragenic recombination at the enn locus and the pattern of distribution of emm and enn alleles among strains suggests that these genes may be independently acquired by horizontal transfer and recombination from distinct donor strains, thereby generating a mosaic structure for the vir regulon. The results add to a growing body of evidence that horizontal gene transfer has played a major role in the evolution of Streptococcus pyogenes vir regulons.
Protein kinase‐dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in Gram‐positive bacteria
Authors: Deutscher Josef , Küster Elke , Bergstedt Uta , Charrier Véronique , Hillen Wolfgang
Published: October 2006

CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram‐positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA‐his immobilized on a Ni‐NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phosphocarrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His‐15 in a PEP‐dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser‐46 in an ATP‐dependent reaction catalysed by a metabolite‐activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His‐15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl‐phosphorylated HPr required the presence of fructose 1,6‐bisphosphate. These findings suggest that carbon catabolite repression in Gram‐positive bacteria is a protein kinase‐triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P‐ser‐HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His‐15 also suggests a link between carbon catabolite repression and PTS transport activity.
L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria
Authors: Sarkis Gary J. , Jacobs William R. , Hatfulll Graham F.
Published: October 2006

Recombinant bacteriophages provide efficient delivery systems for introducing reporter genes into specific bacterial hosts. We have constructed mycobacteriophage L5 recombinants carrying the firefly luciferase gene inserted into the tRNA region of the phage genome. Infection of Mycobacterium smegmatis by these phages results in expression of the luciferase gene and light emission. Fortuitously, the luciferase gene is expressed continuously in lysogens surviving infection. Synthesis of luciferase from a mycobacterial promoter created by cloning enables the detection of extremely small numbers of M. smegmatis cells. These reporter phages can be used to discriminate between drug‐sensitive and drug‐resistant strains of M. smegmatis, and may provide tools for the rapid identification and classification of antimycobacterial agents.
Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ
Authors: Sanna M. Germana , Swanson Ronald V. , Bourret Robert B. , Simon Melvin I.
Published: October 2006

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho‐CheY is thought to determine the swimming behaviour of the cell: high levels of phospho‐CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos‐phorylated form of the protein allow counter‐ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho‐CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho‐CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50‐fold and 5‐fold less sensitive to the activity of CheZ than was the wild‐type protein. Nonetheless, the phosphorylation and autodephos‐phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild‐type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn‐23 and Lys‐26 are located on a face of CheY far from the phosphorylation site (Asp‐57), distinct from the previously described site of inter‐action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.
The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex
Authors: van Gijsegem Frédérique , Gough Clare , Zischek Claudine , Niqueux Eric , Arlat Matthieu , Genin Stéphane , Barberis Patrick , German Sylvie , Castello Philippe , Boucher Christian
Published: October 2006

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR‐inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions., Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant‐pathogenic bacteria include proteins encoded by hrp genes. For Hrp‐related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solanacearum, include proteins encoded by fli and fli genes of S. typhimurium, Bacillus subtils and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.