bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a genetic determinant responsible for host specificity in Streptococcus thermophilus bacteriophages
Authors: Duplessis Martin , Moineau Sylvain
Published: March 2003

Phage–host interactions remain poorly understood in lactic acid bacteria and essentially in all Gram‐positive bacteria. The aim of this study was to identify the phage genetic determinant (anti‐receptor) involved in the recognition of Streptococcus thermophilus hosts. The complete genomic sequence of the lytic S. thermophilus phage DT1 was determined previously, and bioinformatic analysis indicated that orf18 might be the anti‐receptor gene. The orf18 of six additional S. thermophilus phages was determined (DT2, DT4, MD1, MD2, MD4 and Q5) and compared with the orf18 of DT1. The deduced ORF18 was divided into three domains. The first domain, which contains the N‐terminal part of the protein, was conserved in all seven phages. The second domain was detected in only two phages and flanked by a motif called collagen‐like repeats. The second domain also contained a variable region (VR1). All seven phages had a third domain that consisted of the C‐terminal section of the protein as well as another variable region (VR2). Chimeric DT1 phages were constructed by recombination; a portion of its orf18 was replaced by the corresponding section in orf18 of the phage MD4. All DT1 chimeric phages acquired the host range of phage MD4. Analysis of the orf18 in the chimeric phages revealed that host specificity in phages DT1 and MD4 resulted from VR2. This is the first report on the identification and characterization of a phage gene involved in the host recognition process of Gram‐positive bacteria.
The Mycobacterium tuberculosis ECF sigma factor σE: role in global gene expression and survival in macrophages†
Authors: Manganelli Riccardo , Voskuil Martin I. , Schoolnik Gary K. , Smith Issar
Published: March 2003

In previously published work, we identified three Mycobacterium tuberculosis sigma (σ) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the σ factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild‐type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild‐type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription–polymerase chain reaction (RT–PCR), we started to define the σE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when σE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.
Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function
Authors: Caldon Catherine E. , Yoong Pauline , March Paul E.
Published: March 2003

The GTPases comprise a protein superfamily of highly conserved molecular switches adapted to many diverse functions. These proteins are found in all domains of life and often perform essential roles in fundamental cellular processes. Analysis of data from genome sequencing projects demonstrates that bacteria possess a core of 11 universally conserved GTPases (elongation factor G and Tu, initiation factor 2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of GTPases indicate that a second conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes. An emerging concept suggests that the 11 universal GTPases are either necessary for ribosome function or transmitting information from the ribosome to downstream targets for the purpose of generating specific cellular responses. Furthermore, it is suggested that progenitor GTPases were early regulators of RNA function and may have existed in precursors of cellular systems driven by catalytic RNA. If this is the case, then a corollary of this hypothesis is that GTPases that do not bind RNA arose at a later time from an RNA‐binding progenitor that lost the capability to bind RNA.
The fla gene cluster is involved in the biogenesis of flagella in Halobacterium salinarum
Authors: Patenge Nadja , Berendes Antje , Engelhardt Harald , Schuster Stephan C. , Oesterhelt Dieter
Published: March 2003

In this study, a flagella‐related protein gene cluster is described for Halobacterium salinarum. The fla gene cluster is located upstream of the flagellin genes flgB1–3 and oriented in the opposite direction. It consists of nine open reading frames (ORFs): htpIX, a member of the halobacterial transducer protein gene family, and the genes flaD–K. The genes flaD, E, G, H, I and J share high homologies with genes from other Archaea. Interestingly, flaK shows similarities to bacterial genes involved in the regulation of flagellar synthesis. The ORFs of flaH, flaI and flaK contain sequences coding for nucleotide binding sites. Furthermore, flaI contains a motif called the bacterial type II secretion protein E signature, indicating a functional relation to members of the bacterial pili type IV–type II secretion protein superfamily. Reverse transcription–polymerase chain reaction (RT–PCR) analysis revealed that the genes flaE to flaK are transcribed into one polycistronic message. In frame deletion mutants of flaI were generated by gene replacement. The deletion strain lacks motility and belongs to the fla– mutant class, indicating that it is deficient in flagellar biogenesis. The overall amount of flagellin protein in ΔflaI cells is reduced, although transcription of the flagellin genes is unaffected. Therefore, the flaI gene product is involved in the biosynthesis, transport or assembly of flagella in H. salinarum.
The transfer of choline from the host to the bacterial cell surface requires glpQ in Haemophilus influenzae
Authors: Fan Xin , Goldfine Howard , Lysenko Elena , Weiser Jeffrey N.
Published: March 2003

Haemophilus influenzae incorporates choline obtained from environmental sources onto its lipopolysaccharide as phosphorylcholine (ChoP). The decoration of the bacterial surface with ChoP contributes to pathogenesis by allowing for mimicry of the host. As the main reservoir for choline in the host is phosphatidylcholine, we tested whether other choline‐containing molecules associated with eukaryotic membranes could provide an alternative source of choline. H. influenzae was able to use glycerophosphorylcholine (GPC), an abundant degradation product of phospholipids, as efficiently as free choline. Utilization of GPC required glpQ, which expresses an enzyme with glycerophosphodiester phosphodiesterase activity. In the absence of free choline, this gene was required for adherent H. influenzae to obtain choline directly from epithelial cells in culture. GlpQ therefore allows choline to be transferred from the host to the bacterial cell surface.
The pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the peptidyl transferase centre on the ribosome
Authors: Poulsen Susan M. , Karlsson Märit , Johansson Lena B. , Vester Birte
Published: March 2003

The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of peptidyl transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058–9, U2506 and U2584–5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the peptidyl transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a peptidyl transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the peptidyl transferase slot on the ribosomes in which they prevent the correct positioning of the CCA‐ends of tRNAs for peptide transfer.
Mutation of luxS affects growth and virulence factor expression in Streptococcus pyogenes
Authors: Lyon William R. , Madden John C. , Levin James C. , Stein Jeffrey L. , Caparon Michael G.
Published: March 2003

Adaptive responses of bacteria that involve sensing the presence of other bacteria are often critical for proliferation and the expression of virulence characteristics. The autoinducer II (AI‐2) pathway has recently been shown to be a mechanism for sensing other bacteria that is highly conserved among diverse bacterial species, including Gram‐positive pathogens. However, a role for this pathway in the regulation of virulence factors in Gram‐positive pathogens has yet to be established. In this study, we have inactivated luxS, an essential component of the AI‐2 pathway, in the Gram‐positive pathogen Streptococcus pyogenes. Analyses of the resulting mutants revealed the aberrant expression of several virulence properties that are regulated in response to growth phase, including enhanced haemolytic activity, and a dramatic reduction in the expression of secreted proteolytic activity. This latter defect was associated with a reduced ability to secrete and process the precursor of the cysteine protease (SpeB) as well as a difference in the timing of expression of the protease. Enhanced haemolytic activity of the luxS strain was also shown to be linked with an increased expression of the haemolysin S‐associated gene sagA. Disruptions of luxS in these mutants also produced a media‐dependent growth defect. Finally, an allelic replacement analysis of an S. pyogenes strain with a naturally occurring insertion of IS1239 in luxS suggested a mechanism for modulation of virulence during infection. Results from this study suggest that luxS makes an important contribution to the regulation of S. pyogenes virulence factors.
Phosphorylation of tyrosine 972 of the Helicobacter pylori CagA protein is essential for induction of a scattering phenotype in gastric epithelial cells
Authors: Backert Steffen , Moese Stefan , Selbach Matthias , Brinkmann Volker , Meyer Thomas F.
Published: March 2003

Helicobacter pylori colonizes the human stomach and is the causative agent of a variety of gastric diseases. After bacterial attachment, the H. pylori CagA protein is translocated into gastric epithelial cells and tyrosine phosphorylated. This process is associated with characteristic cytoskeletal rearrangements, resulting in a scatter factor‐like (‘hummingbird’) phenotype. In this study, using a cagA mutant complemented with wild‐type cagA and transiently expressing CagA in AGS cells, we have demonstrated that translocated CagA is necessary for rearrangements of the actin cytoskeleton to occur. Anti‐phosphotyrosine immunoblotting studies and treatment of infected cells with phosphotyrosine kinase inhibitors suggested that not only translocation but also phosphorylation of CagA is important in this process. Transient expression of CagA–green fluorescent protein (GFP) fusion proteins and two‐dimensional gel electrophoresis of CagA protein species demonstrated tyrosine phosphorylation in the C‐terminus. Site‐directed mutagenesis of CagA revealed that tyrosine residue 972 is essential for induction of the cellular phenotype. We have also demonstrated that translocation and phosphorylation of CagA is necessary but not sufficient for induction of the hummingbird phenotype in AGS cells, indicating the involvement of as yet unidentified bacterial factor(s).
A secondary RNA polymerase sigma factor from Streptococcus pyogenes
Authors: Opdyke Jason A. , Scott June R. , Moran Charles P.
Published: March 2003

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) causes diseases ranging from mild, self‐limiting pharyngitis to severe invasive infections. Regulation of the expression of GAS genes in response to specific environmental differences within the host is probably key in determining the course of the infectious process, however, little is known of global regulators of gene expression in GAS. Although secondary RNA polymerase sigma factors act as global regulators of gene expression in many other bacteria, none has yet been isolated from the GAS. The newly available GAS genome sequence indicates that the only candidate secondary sigma factor is encoded by two identical open reading frames (ORFS). These ORFS encode a protein that is 40% identical to the transcription factor ComX, believed to act as an RNA polymerase sigma factor in Streptococcus pneumoniae. To test whether the GAS ComX homologue functions as a sigma factor, we cloned and purified it from Escherichia coli. We found that in vitro, this GAS protein, which we call σX, directed core RNA polymerase from Bacillus subtilis to transcribe from two GAS promoters that contain the cin‐box region, required for transcription by S. pneumoniae ComX in vivo. On the other hand, GAS σX did not promote transcription of a GAS promoter (hasA) expected to be dependent on σA, the housekeeping or primary RNA polymerase sigma factor. Addition of monoclonal antibody that inhibited σA‐directed transcription had no effect on σX‐directed transcription, showing that the latter was not the result of contaminating σA. Transcription of both cin‐box‐containing promoters initiated downstream of the cin‐box and two different single basepair substitutions in the cin‐box of the cinA promoter each caused a severe reduction of σX‐directed transcription in vitro. Thus, the cin‐box is required for σX‐directed transcription.
Light‐induced carotenogenesis inMyxococcus xanthus: evidence that CarS acts as an anti‐repressor of CarA
Authors: Whitworth David E. , Hodgson David A.
Published: March 2003

In the bacteriumMyxococcus xanthus, carotenoids are produced in response to illumination, as a result of expression of thecrtcarotenoid biosynthesis genes. The majority ofcrtgenes are clustered in thecrtEBDCoperon, which is repressed in the dark by CarA. Genetic data suggest that, in the light, CarS is synthesized and achieves activation of thecrtEBDCoperon by removing the repressive action of CarA. As CarS contains no known DNA‐binding motif, the relief of CarA‐mediated repression was postulated to result from a direct interaction between these two proteins. Use of the yeast two‐hybrid system demonstrated direct interaction between CarA and CarS. The two‐hybrid system also implied that CarA and, possibly, CarS are capable of homodimerization. Direct evidence for CarS anti‐repressor action was providedin vitro. A glutathioneS‐transferase (GST)–CarA protein fusion was shown to bind specifically to a palindromic operator sequence within thecrtEBDCpromoter. CarA was prevented from binding to its operator, and prebound CarA was removed by the addition of purified CarS. CarS is therefore an anti‐repressor.
Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in morphogenesis and growth of Candida albicans
Authors: Bockmühl Dirk P. , Krishnamurthy Shankarling , Gerads Michaela , Sonneborn Anja , Ernst Joachim F.
Published: March 2003

TPK1 and TPK2 encode both isoforms of protein kinase A (PKA) catalytic subunits in Candida albicans. Mutants lacking both TPK1 alleles showed defective hyphal morphogenesis on solid inducing media, whereas in liquid hypha, formation was affected slightly. In contrast, tpk2 mutants were only partially morphogenesis defective on solid media, whereas a strong block was observed in liquid. In addition, the yeast forms of tpk2– but not tpk1– mutants were completely deficient in invading agar. Because Tpk1p and Tpk2p differ in their N‐terminal domains of approximately 80–90 amino acids, while the catalytic portions are highly homologous, the functions of hybrid Tpk proteins with exchanged N‐terminal domains were tested. The results demonstrate that the catalytic portions mediate Tpk protein specificities with regard to filamentation, whereas agar invasion is mediated by the N‐terminal domain of Tpk2p. Homozygous tpk1 and tpk2 mutants grew normally; however, a tpk2 mutant strain containing a single regulatable TPK1 allele (PCK1p‐TPK1) at low expression levels was severely growth defective. It was completely blocked in hyphal morphogenesis and was stress resistant to high osmolarities or temperatures. Thus, both Tpk isoforms in C. albicans share growth functions but, unlike Saccharomyces cerevisiae isoforms, they have positive, specific roles in filament formation in different environments.
The type III secretion chaperone LcrH co‐operates with YopD to establish a negative, regulatory loop for control of Yop synthesis in Yersinia pseudotuberculosis
Authors: Francis Matthew S. , Lloyd Scott A. , Wolf‐Watz Hans
Published: March 2003

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram‐negative pathogens secrete and subsequently translocate antihost effector proteins into target eukaryotic cells by a common type III secretion system (TTSS). In this process, YopD (Yersiniaouter protein D) is essential to establish regulatory control of Yop synthesis and the ensuing translocation process. YopD function depends upon the non‐secreted TTSS chaperone LcrH (low‐calcium response H), which is required for presecretory stabilization of YopD. However, as a new role for TTSS chaperones in virulence gene regulation has been proposed recently, we undertook a detailed analysis of LcrH. A lcrH null mutant constitutively produced Yops, even when this strain was engineered to produce wild‐type levels of YopD. Furthermore, the YopD–LcrH interaction was necessary to regain the negative regulation of virulence associated genes yops). This finding was used to investigate the biological significance of several LcrH mutants with varied YopD binding potential. Mutated LcrH alleles were introduced in trans into a lcrH null mutant to assess their impact on yop regulation and the subsequent translocation of YopE, a Rho‐GTPase activating protein, across the plasma membrane of eukaryotic cells. Two mutants, LcrHK20E, E30G, I31V, M99V, D136G and LcrHE30G lost all regulatory control, even though YopD binding and secretion and the subsequent translocation of YopE was indistinguishable from wild type. Moreover, these regulatory deficient mutants showed a reduced ability to bind YscY in the two‐hybrid assay. Collectively, these findings confirm that LcrH plays an active role in yop regulation that might be mediated via an interaction with the Ysc secretion apparatus. This chaperone–substrate interaction presents an innovative means to establish a regulatory hierarchy in Yersinia infections. It also raises the question as to whether or not LcrH is a true chaperone involved in stabilization and secretion of YopD or a regulatory protein responsible for co‐ordinating synthesis of Yersinia virulence determinants. We suggest that LcrH can exhibit both of these activities.
Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins
Authors: Shimizu Tohru , Yaguchi Harumi , Ohtani Kaori , Banu Sayera , Hayashi Hideo
Published: March 2003

We analysed the region encoding VR‐RNA (VirR‐regulated RNA), which has been reported to be positively regulated by the two‐component VirR/VirS system in Clostridium perfringens. The VR‐RNA promoter identified by primer extension analysis was preceded by a probable VirR‐binding site (CCAGTTNNNCAC), which resembled a repeated sequence motif present in the promoter region of the theta‐toxin (pfoA) gene. A VR‐RNA‐null mutant, constructed by a homologous recombination, exhibited a reduced amount of transcription of the alpha‐ (plc) and kappa‐toxin (colA) genes, which was restored by the complementation of intact VR‐RNA, indicating that the VR‐RNA region plays an important role in the regulation of the plc and colA genes in C. perfringens. It was found that the regulatory effect was observed even when the hyp7 gene encoded on VR‐RNA was deleted or a nonsense mutation was introduced in the hyp7‐coding region. We found that the small 3′‐portion of VR‐RNA was sufficient for the activation of toxin genes, which suggested that VR‐RNA itself could act as an RNA regulatory molecule for the plc and colA genes mediating the regulatory information from the VirR/VirS system in C. perfringens.
Analysis of the kefA2 mutation suggests that KefA is a cation‐specific channel involved in osmotic adaptation in Escherichia coli
Authors: McLaggan Debra , Jones Michael A. , Gouesbet Gwenola , Levina Natasha , Lindey Susanna , Epstein Wolfgang , Booth Ian R.
Published: March 2003

Mechanosensitive channels play an essential role in the regulation of turgor pressure in bacteria. In Escherichia coli, there are multiple mechanosensitive channels that have been characterized genetically: MscL, YggB and KefA. In this report, we describe the cloning of the kefA gene, the organization of the KefA protein and the phenotype of a missense mutation, kefA, which affects the KefA mechanosensitive channel. The altered function of the channel is manifest through increased sensitivity to K+ during growth at low osmolarity and complete inhibition of growth in media containing high K+ concentrations (0.6 M) in the presence of betaine or proline. Growth in high Na+ medium (0.6 M NaCl plus 20 mM K+) is normal. Analysis of the cytoplasmic pools shows that the mutant cannot regulate the K+ content of the cytoplasm when grown in high K+ medium. However, regulation of pools of amino acids is essentially normal and the mutant can accumulate high pools of proline during growth inhibition. The mutant shows increased sensitivity to acid hypo‐osmotic shock (transition from neutral to acid pH combined with a reduction in osmolarity). The data are consistent with abnormal regulation of KefA in the presence of high K+ concentrations and either betaine or proline.
Functional genomics reveals the sole sulphate transporter of the Mycobacterium tuberculosis complex and its relevance to the acquisition of sulphur in vivo
Authors: Wooff Esen , Michell Stephen Ll. , Gordon Stephen V. , Chambers Mark A. , Bardarov Stoyan , Jacobs William R. , Hewinson R. Glyn , Wheeler Paul R.
Published: March 2003

Sulphur is essential for some of the most vital biological activities such as translation initiation and redox maintenance, and genes involved in sulphur metabolism have been implicated in virulence. Mycobacterium tuberculosis has three predicted genes for the prototrophic acquisition of sulphur as sulphate: cysA, part of an ABC transporter, and cysA2 and A3, SseC sulphotransferases. Screening for amino acid auxotrophs of Mycobacterium bovis BCG, obtained by transposon mutagenesis, was used to select methionine auxotrophs requiring a sulphur‐containing amino acid for growth. We have characterized one of these auxotrophs as being disrupted in cysA. Both the cysA mutant and a previously identified mutant in an upstream gene, subI, were functionally characterized as being completely unable to take up sulphate. Complementation of the cysA mutant with the wild‐type gene from M. tuberculosis restored prototrophy and the ability to take up sulphate with the functional characteristics of an ABC transporter. Hence, it appears that this is the sole locus encoding inorganic sulphur transport in the M. tuberculosis complex
The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase
Authors: Pisa René , Stein Torsten , Eichler Robert , Gross Roland , Simon Jörg
Published: March 2003

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem‐binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenesΔnrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix‐assisted laser‐desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild‐type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild‐type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.
Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion
Authors: Brayton Kelly A. , Palmer Guy H. , Lundgren Anna , Yi Jooyoung , Barbet Anthony F.
Published: March 2003

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent
infection in the mammalian reservoir host, during which time immune escape variants
continually arise in part because of variation in the expressed copy of the immunodominant
outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale
genome generates sufficient variants to allow long‐term persistence in an immunocompetent
reservoir host. The recombination of whole pseudogenes into the single msp2
expression site has been previously identified as one method of generating variants,
but is inadequate to generate the number of variants required for persistent infection.
In the present study, we demonstrate that recombination of a whole pseudogene is
followed by a second level of variation in which small segments of pseudogenes recombine
into the expression site by gene conversion. Evidence for four short sequential changes
in the hypervariable region of msp2 coupled with the identification of nine
pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.
ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae
Authors: Li Caiyi C. , Merrell D. Scott , Camilli Andrew , Kaper James B.
Published: March 2003

In pathogenic Vibrio cholerae, the transmembrane DNA‐binding protein ToxR

co‐ordinates the expression of over 20 genes, including those encoding important

virulence factors such as cholera toxin and the toxin‐co‐regulated pilus. The outer

membrane protein OmpT is the only member of the ToxR regulon known to be repressed

by ToxR. In this study, we examined the environmental conditions that regulate OmpT

expression and demonstrated that ompT transcription is upregulated 14‐fold

when the bacteria enter late log phase from early log phase. Deletion of the crp

gene completely abolishes OmpT expression. Comparison of ompT transcription

levels in the isogenic crp−, toxR−

and crp−toxR− mutants

revealed that (i) in the absence of ToxR, constitutive high‐level ompT transcription

is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP‐dependent

ompT activation, but also abolishes the CRP‐independent, basal level ompT

transcription; thus, the mechanism by which ToxR represses ompT transcription

involves both antiactivation and direct repression; (iii) both CRP and ToxR are required

for the regulation of OmpT expression by growth phase. To provide further insights

into the molecular mecha‐nism of CRP‐dependent activation of ompT transcription,

we demonstrated that CRP‐dependent activation requires a CRP binding site centred

at −310 of the ompT promoter, without which the interaction of

CRP with other CRP binding site(s) more proximal to the promoter results in repression.

Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase

(RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from −310

of the ompT promoter via DNA looping.
RNase G complementation of rne null mutation identifies functional interrelationships with RNase E in Escherichia coli
Authors: Lee Kangseok , Bernstein Jonathan A. , Cohen Stanley N.
Published: March 2003

The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have

sequence similarity and broadly similar sequence specificity. Whereas the absence

of Rne normally is lethal, we show here that E. coli bacteria that lack the

rne gene can be made viable by overexpression of Rng. Rng‐complemented cells

accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase

P (i.e. M1 RNA), indicating that normal processing of these Rne‐cleaved RNAs was

not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from

precursors by RNase G cleavage in vitro. Using DNA microarrays containing

4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose

steady‐state level was affected by Rne, Rng or the N‐terminal catalytic domain of

RNase E. Most transcript species affected by RNase E deficiency were also elevated

in an rne deletion mutant complemented by Rng. However, approximately 100

mRNAs that accumulated in Rne‐deficient cells were decreased by rng‐complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy‐generating pathways or in the synthesis or degradation of macromolecules.
Regulation of autolysins in teichuronic acid‐containingBacillus subtiliscells
Authors: Calamita Heather G. , Doyle R. J.
Published: March 2003

Bacillus subtiliscells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA). The turnover rates of TA‐containing and TUA‐containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar. In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)–dextran to probe the wall pH, that a low pH exists in the wall matrix. A second probe, cationized ferritin (CF), was used to observe cell surface protonation. Suspensions ofB. subtiliscells containing either TA or TUA were aggregated with CF only after the addition of a protonmotive force‐dissipating agent. RespiringB. subtilisTUA‐containing cells labelled with FITC–dextran exhibited little fluorescence. Conversely, fluorescence intensities exhibited by cells de‐energized with nitrogen gas were significantly greater. The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA‐containing and TUA‐containingB. subtiliscells. Both TUA‐containing and TA‐containing cells were lysed only after the addition of sodium azide. These data suggest that during normal growth the wall of TUA‐containingB.subtilis cells is protonated, and protonmotive force influences autolytic regulation in both TUA‐containing and TA‐containing B. subtilis cells.
Binding site requirements of the virulence gene regulator AphB: differential affinities for the Vibrio cholerae classical and El Tor tcpPH promoters
Authors: Kovacikova Gabriela , Skorupski Karen
Published: March 2003

The differential expression of virulence genes be‐tween the two disease‐causing biotypes of Vibrio cholerae, classical and El Tor, is primarily due to a single basepair change in the tcpPH promoter, which strongly influences the ability of the LysR regulator AphB to activate transcription in response to environmental conditions. We show here that this single basepair change influences virulence gene expression by dramatically altering the affinity of AphB for its recognition site in the tcpPH promoter. AphB binds greater than 10‐fold more efficiently to a wild‐type classical tcpPH promoter fragment with an A at −65 relative to a wild‐type El Tor fragment that has a G at this position. As this single basepair change is located within the left arm of the LysR recognition motif (5′‐TGCAA‐N7‐TTGCA), which extends from −69 to −53, a systematic mutagenesis of the other positions within this site was carried out to assess their influence on AphB binding in vitro and transcriptional activation in vivo. This analysis revealed that the left and right arms of the interrupted dyad display a high degree of symmetry with respect to their role in AphB binding. The right promoter proximal arm also plays a role in transcriptional activation that is distinct from its role in AphB binding. A second AphB binding site (5′‐TGCAA‐N7‐TGTCA) was identified upstream of the aphB gene itself, which extends from +17 to +33 relative to the start of transcription and functions in autorepression. Although the sequences of the AphB binding sites at the tcpPH and aphB promoters are highly conserved, important differences exist in the way that AphB functions at each of these sites.
The cyanobacterial phytochrome Cph2 inhibits phototaxis towards blue light
Authors: Wilde Annegret , Fiedler Brita , Börner Thomas
Published: March 2003

We analysed the effects of inactivation of the phytochrome genes cph1 and
cph2 on the phototactic migration of the cyanobacterium Synechocystis
sp. PCC 6803 under different light qualities. Motility towards white, green, red
and far red light was not altered in several independently raised cph1, cph2
and cph1/cph2 double mutants. Blue light (λ = 400–500
nm) did not induce motility in wild‐type and cph1 mutant cells, whereas cph2
and cph1/cph2 double mutants moved towards the blue light. Inhibition
of the photosynthetic electron transport by DCMU influenced the motility of cph2
mutants under blue light, but not under white, red and far red light. RNA blot hybridizations
did not indicate an altered transcript accumulation of the pilin‐encoding pilA1
gene under blue light. We propose that the Cph2 protein is part of a light‐stimulated
signal transduction chain inhibiting the movement of Synechocystis sp. PCC 6803 cells towards blue light.
Differential gene expression in Entamoeba histolytica isolated from amoebic liver abscess
Authors: Bruchhaus Iris , Roeder Thomas , Lotter Hannelore , Schwerdtfeger Maike , Tannich Egbert
Published: March 2003

The majority of human infections with the intestinal protozoan parasite Entamoeba histolytica remain asymptomatic. In a small proportion of infections, however, E. histolytica trophozoites penetrate the intestinal mucosa and disseminate to other organs, most commonly to the liver, where they induce abscess formation. It is believed that the ability of E. histolytica trophozoites to destroy host tissues and to survive within the liver is accomplished by a strong adaptive response, which requires the specific regulation of a number of amoeba proteins. Using differential display polymerase chain reaction (DD‐PCR), we compared RNA expression between E. histolytica trophozoites isolated from liver abscesses of infected gerbils and those grown under normal culture conditions. A total of 3000 cDNA‐derived amplicons were compared between the two groups of amoebae, which were calculated to represent about one‐third of all E. histolytica mRNA species (transcriptome). Among these, 55 were found to be specifically present or absent in abscess‐derived amoebae, of which 42 were successfully cloned and sequenced. Database searches and Northern blot analyses revealed that the 42 amplicons correspond to 29 independent E. histolytica genes, of which at least seven are specifically upregulated and five are downregulated in abscess‐derived amoebae. Specific expression of most of these genes was not simply the result of a heat shock response, which might be expected during abscess formation, as only five of the genes revealed an expression profile similar to that found in amoebae cultured under elevated temperatures. The two genes specifically downregulated in abscess‐derived amoebae encode members of a family of so far unknown proteins, which contain repetitive stretches of sequences that are rich in lysine and glutamic acid residues. In contrast, a diverse set of genes is specifically upregulated, encoding ribosomal proteins (S30, L37A), cyclophilin, ferredoxin 2 and GTP‐binding protein RAB7D, supporting the notion that liver abscess formation requires the regu‐lation and concerted action of a variety of amoeba proteins. These proteins are associated with stress response, signal transduction, regulation of transcription and vesicular trafficking. However, transcriptome analysis will not be sufficient to identify all proteins specifically upregulated during abscess formation, as at least an increase in the expression of actin was found to be regulated at the post‐transcriptional level.
A novel cyclic GMP‐dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle
Authors: Deng Wensheng , Baker David A.
Published: March 2003

Cyclic GMP‐dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. We report here the characterization of an unusual PKG from the human malaria parasite Plasmodium
falciparum (designated PfPKG). The 97.5 kDa protein contains some of the structural
features of mammalian PKGs but, uniquely, contains a third predicted cGMP binding
site and a degenerate fourth. Using both protein kinase activity assays and Western
blotting with native P. falciparum proteins, we demonstrate here that PfPKG
is expressed predominantly in the ring stage of the life cycle, suggesting a role
in the development of asexual blood stage parasites. An Escherichia coli‐derived
recombinant protein (PfPKG2, Met115‐Phe853) was purified and shown to have phosphotransferase activity in terms of both substrate phosphorylation and auto‐phosphorylation. This activity was stimulated at least fivefold by 1.0 μ M cyclic GMP, but was not stimulated by cAMP or by 8‐pCPT‐cGMP, which is a potent activator of mammalian PKGs. Several protein kinase inhibitors exhibited a range of inhibitory effects on PfPKG activity. Biochemical analysis therefore shows that PfPKG is distinct from mammalian PKGs with respect to both cyclic nucleotide analogue activation and inhibition profiles.
The ShcA protein is a molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein secretion system of Pseudomonas syringae
Authors: Dijk Karin van , Tam Vincent C. , Records Angela R. , Petnicki‐Ocwieja Tanja , Alfano James R.
Published: March 2003

Pseudomonas syringae uses a type III protein secretion system encoded by
the Hrp pathogenicity island (Pai) to translocate effector proteins into plant cells.
One of these effector proteins is HopPsyA. A small open reading frame (ORF), named
shcA, precedes the hopPsyA gene in the Hrp Pai of P. s. syringae
61. The predicted amino acid sequence of shcA shares general characteristics
with chaperones used in type III protein secretion systems of animal pathogens. A
functionally non‐polar deletion of shcA in P. s. syringae 61 resulted
in the loss of detectable HopPsyA in supernatant fractions, consistent with ShcA
acting as a chaperone for HopPsyA. Cosmid pHIR11 carries a functional set of type
III genes from P. s. syringae 61 and confers upon saprophytes the ability
to secrete HopPsyA in culture and to elicit a HopPsyA‐dependent hypersensitive response
(HR) on tobacco. P. fluorescens carrying a pHIR11 derivative lacking shcA
failed to secrete HopPsyA in culture, but maintained the ability to secrete another
type III‐secreted protein, HrpZ. This pHIR11 derivative was also greatly reduced
in its ability to elicit an HR, indicating that the ability to translocate HopPsyA
into plant cells was compromised. Using affinity chromatography, we showed that ShcA
binds directly to HopPsyA and that the ShcA binding site must reside within the first
166 amino acids of HopPsyA. Thus, ShcA represents the first demonstrated chaperone
used in a type III secretion system of a bacterial plant pathogen. We searched known
P. syringae type III‐related genes for neighbouring ORFs that shared the general characteristics of type III chaperones and identified five additional candidate type III chaperones. Therefore, it is likely that chaperones are as prevalent in bacterial plant pathogen type III systems as they are in their animal pathogenic counterparts.
Genomics of the marA/soxS/rob regulon of Escherichia coli: identification of directly activated promoters by application of molecular genetics and informatics to microarray data
Authors: Martin Robert G. , Rosner Judah L.
Published: March 2003

Microarray analyses are providing a plethora of data concerning transcriptional responses to specific gene regulators and their inducers but do not distinguish between direct and indirect responses. Here, we identify directly activated promoters of the overlapping marA, soxS and rob regulon(s) of Escherichia coli by applying informatics, genomics and molecular genetics to microarray data obtained by others. Those studies found that overexpression of marA, or the treatment of cells with salicylate to derepress marA, or treatment with paraquat to induce soxS, resulted in elevated transcription of 153 genes. However, only 27 out of the promoters showed increased transcription under at least two of the aforementioned conditions and eight of those were previously known to be directly activated. A computer algorithm was used to identify potential activator binding sites located upstream of the remaining 19 promoters of this subset, and conventional genetic and biochemical approaches were applied to test whether these sites are critical for activation by the homologous MarA, SoxS and Rob transcriptional activators. Only seven out of the 19 promoters were found to be activated when fused to lacZ and tested as single lysogens. All seven contained an essential activator binding site. The remaining promoters were insensitive to stimulation by the inducers suggesting that the great majority of elevated microarray transcripts either were misidentified or resulted from indirect effects requiring sequences outside of the promoter region. We estimate that the total number of directly activated promoters in the regulon is less than 40.
The autoregulatory role of EsaR, a quorum‐sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function
Authors: Minogue Timothy D. , Trebra Markus Wehland‐von , Bernhard Frank , Bodman Susanne B. von
Published: March 2003

Capsular polysaccharide synthesis and virulence in the plant pathogenic bacterium Pantoea stewartii ssp. stewartii requires the quorum‐sensing regulatory proteins, EsaR and EsaI, and the diffusible inducer N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone. Prior mu‐tational studies suggested that EsaR might function as a repressor of quorum sensing in the control of capsular polysaccharide synthesis. Further, a lux box‐like palindromic sequence coinciding with the putative –10 element of the esaR promoter suggested a possible negative autoregulatory role for EsaR. This report presents genetic evidence that EsaR represses the esaR gene under inducer‐limiting conditions, and that addition of inducer promotes rapid, dose‐dependent derepression. DNA mobility‐shift assays and analyses by surface plasmon resonance refractometry show that EsaR binds target DNAs in a ligand‐free state, and that inducer alters the binding characteristics of EsaR. Physical measurements indicate that the EsaR protein binds N‐(3‐oxo‐hexanoyl)‐L‐homoserine lactone, in a 1:1 protein:ligand ratio, and that inducer binding enhances the thermal stability of the EsaR protein. These combined genetic and biochemical data establish that EsaR regulates its own expression by signal‐independent repression and signal‐dependent derepression. Additionally, we provide evidence that EsaR does not govern the expression of the linked esaI gene, thus EsaR has no role in controlling coinducer synthesis.
An antiport mechanism for a member of the cation diffusion facilitator family: divalent cations efflux in exchange for K+ and H+
Authors: Guffanti Arthur A. , Wei Yi , Rood Sacha V. , Krulwich Terry A.
Published: March 2003

Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cad‐mium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.