bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Architecture of the Gram-Negative Bacterial Murein Sacculus
Authors: Pink David , Moeller Jeremy , Quinn Bonnie , Jericho Manfred , Beveridge Terry
Published: July 2002

The peptidoglycan network of the murein sacculus must be porous so that nutrients, waste products, and secreted proteins can pass through. Using
Escherichia coli
and
Pseudomonas aeruginosa
as a baseline for gram-negative sacculi, the hole size distribution in the peptidoglycan network has been modeled by computer simulation to deduce the network's properties. By requiring that the distribution of glycan chain lengths predicted by the model be in accord with the distribution observed, we conclude that the holes are slits running essentially perpendicular to the local axis of the glycan chains (i.e., the slits run along the long axis of the cell). This result is in accord with previous permeability measurements of Beveridge and Jack and Demchik and Koch. We outline possible advantages that might accrue to the bacterium via this architecture and suggest ways in which such defect structures might be detected. Certainly, large molecules do penetrate the peptidoglycan layer of gram-negative bacteria, and the small slits that we suggest might be made larger by the bacterium.
Characterization of the DNA- and Metal-Binding Properties of
Vibrio anguillarum
Fur Reveals Conservation of a Structural Zn
2+
Ion
Authors: Zheleznova Ekaterina E. , Crosa Jorge H. , Brennan Richard G.
Published: July 2002

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on
Vibrio anguillarum
Fur binding to the consensus 19-bp
Escherichia coli
iron box in the presence of several divalent metals. We found that
V. anguillarum
Fur binds the iron box in the presence of Mn
2+
, Co
2+
, Cd
2+
, and to a lesser extent Ni
2+
but, unlike
E. coli
Fur, not in the presence of Zn
2+
. We also found that
V. anguillarum
Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.
N
-Acetyl-1-
d
-
myo
-Inosityl-2-Amino-2-Deoxy-α-
d
-Glucopyranoside Deacetylase (MshB) Is a Key Enzyme in Mycothiol Biosynthesis
Authors: Newton Gerald L. , Av-gay Yossef , Fahey Robert C.
Published: July 2002

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria. Mycothiol was previously shown to be synthesized from 1-
d
-
myo
-inosityl-2-amino-2-deoxy-α-
d
-glucopyranoside by ligation with cysteine followed by acetylation. A novel mycothiol-dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in
Mycobacterium smegmatis
and shown to have a homolog, Rv1082, in
Mycobacterium tuberculosis
. In the present study we found that a protein encoded by the
M. tuberculosis
open reading frame Rv1170, a homolog of Rv1082, possesses weak mycothiol conjugate amidase activity but shows substantial deacetylation activity with 1-
d
-
myo
-inosityl-2-acetamido-2-deoxy-α-
d
-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic precursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in
M. smegmatis
at a level about twice that of mycothiol. It was shown that GlcNAc-Ins is absent in mycothiol-deficient mutant strain 49 of
M. smegmatis
and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol. This demonstrates that GlcNAc-Ins is a key intermediate in the pathway of mycothiol biosynthesis. Assignment of Rv1170 as the gene coding the deacetylase in the
M. tuberculosis
genome represents the first identification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.
Proteome Analysis of the Effect of Mucoid Conversion on Global Protein Expression in
Pseudomonas aeruginosa
Strain PAO1 Shows Induction of the Disulfide Bond Isomerase, DsbA
Authors: Malhotra Sonal , Silo-Suh Laura A. , Mathee Kalai , Ohman Dennis E.
Published: July 2002

Pseudomonas aeruginosa
strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its
mucA22
derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by
mucA
mutation. Two other protein spots were also clearly upregulated in the
mucA22
background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The
oprF-lacZ
fusion showed little difference in levels of expression in the two strains. However, the
dsbA-lacZ
fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A
dsbA
-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the
dsbA
mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of
dsbA
in the mucoid PDO300 background did not affect alginate production. Thus, even though
dsbA
is coregulated with mucoid conversion, it was not required for alginate production. This suggests that
mucA
mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.
pING Family of Conjugative Plasmids from the Extremely Thermophilic Archaeon
Sulfolobus islandicus
: Insights into Recombination and Conjugation in Crenarchaeota
Authors: Stedman Kenneth M. , She Qunxin , Phan Hien , Holz Ingelore , Singh Harpreet , Prangishvili David , Garrett Roger , Zillig Wolfram
Published: July 2002

A novel family of conjugative plasmids from
Sulfolobus
comprising the active variants pING1, -4, and -6 and the functionally defective variants pING2 and -3, which require the help of an active variant for spreading, has been extensively characterized both functionally and molecularly. In view of the sparse similarity between bacterial and archaeal conjugation and the lack of a practical genetic system for
Sulfolobus
, we compared the functions and sequences of these variants and the previously described archaeal conjugative plasmid pNOB8 in order to identify open reading frames (ORFs) and DNA sequences that are involved in conjugative transfer and maintenance of these plasmids in
Sulfolobus
. The variants pING4 and -6 are reproducibly derived from pING1 in vivo by successive transpositions of an element from the
Sulfolobus
genome. The small defective but mobile variants pING2 and -3, which both lack a cluster of highly conserved ORFs probably involved in plasmid transfer, were shown to be formed in vivo by recombinative deletion of the larger part of the genomes of pING4 and pING6, respectively. The efficient occurrence of these recombination processes is further evidence for the striking plasticity of the
Sulfolobus
genome.
Purification and Characterization of a Catalase from the Facultatively Psychrophilic Bacterium
Vibrio rumoiensis
S-1
T
Exhibiting High Catalase Activity
Authors: Yumoto Isao , Ichihashi Daisen , Iwata Hideaki , Istokovics Anita , Ichise Nobutoshi , Matsuyama Hidetoshi , Okuyama Hidetoshi , Kawasaki Kosei
Published: July 2002

Catalase from the facultatively psychrophilic bacterium
Vibrio rumoiensis
S-1
T
, which was isolated from an environment exposed to H
2
O
2
and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U · mg of protein
−1
under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of the
Micrococcus luteus
and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1
T
is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1
T
catalase and
M. luteus
and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1
T
should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1
T
is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.
Role of Trehalose in Growth at High Temperature of
Salmonella enterica
Serovar Typhimurium
Authors: Cánovas David , Fletcher Susanne A. , Hayashi Mikachi , Csonka Laszlo N.
Published: July 2002

Moderate osmolality can stimulate bacterial growth at temperatures near the upper limit for growth. We investigated the mechanism by which high osmolality enhances the thermotolerance of
Salmonella enterica
serovar Typhimurium, by isolating bacteriophage MudI1734-induced insertion mutations that blocked the growth-stimulatory effect of 0.2 M NaCl at 45°C. One of these mutations proved to be in the
seqA
gene (a regulator of initiation of DNA synthesis). Because this gene is cotranscribed with
pgm
(which encodes phosphoglucomutase)
,
it is likely to be polar on the expression of the
pgm
gene. Pgm catalyzes the conversion of glucose-6-phosphate to glucose-1-phosphate during growth on glucose, and therefore loss of Pgm results in a deficiency in a variety of cellular constituents derived from glucose-1-phosphate, including trehalose. To test the possibility that the growth defect of the
seqA
::MudI1734 mutant at high temperature in medium of high osmolality is due to the block in trehalose synthesis, we determined the effect of an
otsA
mutation, which inactivates the first step of the trehalose biosynthetic pathway. The
otsA
mutation caused a growth defect at 45°C in minimal medium containing 0.2 M NaCl that was similar to that caused by the
pgm
mutation, but
otsA
did not affect growth rate in this medium at 37°C. These results suggest that the growth defect of the
seqA
-
pgm
mutant at high temperature could be a consequence of the block in trehalose synthesis. We found that, in addition to the well-known osmotic control, there is a temperature-dependent control of trehalose synthesis such that, in medium containing 0.2 M NaCl, cells grown at 45°C had a fivefold higher trehalose pool size than cells grown at 30°C. Our observations that trehalose accumulation is thermoregulated and that mutations that block trehalose synthesis cause a growth defect at high temperature in media of high osmolality suggested that this disaccharide is crucial for growth at high temperature either for turgor maintenance or for protein stabilization.
The
Bordetella bhu
Locus Is Required for Heme Iron Utilization
Authors: Vanderpool Carin K. , Armstrong Sandra K.
Published: July 2002

Bordetella pertussis
and
Bordetella bronchiseptica
are capable of obtaining iron from hemin and hemoglobin. Genes encoding a putative bacterial heme iron acquisition system (
bhu
, for
Bordetella
heme utilization) were identified in a
B. pertussis
genomic sequence database, and the corresponding DNA was isolated from a virulent strain of
B. pertussis
. A
B. pertussis bhuR
mutant, predicted to lack the heme outer membrane receptor, was generated by allelic exchange. In contrast to the wild-type strain,
bhuR
mutant PM5 was incapable of acquiring iron from hemin and hemoglobin; genetic complementation of PM5 with the cloned
bhuRSTUV
genes restored heme utilization to wild-type levels. In parallel studies,
B. bronchiseptica bhu
sequences were also identified and a
B. bronchiseptica bhuR
mutant was constructed and confirmed to be defective in heme iron acquisition. The wild-type
B. bronchiseptica
parent strain grown under low-iron conditions produced the presumptive BhuR protein, which was absent in the
bhuR
mutant. Furthermore, production of BhuR by iron-starved
B. bronchiseptica
was markedly enhanced by culture in hemin-supplemented medium, suggesting that these organisms sense and respond to heme in the environment. Analysis of the genetic region upstream of the
bhu
cluster identified open reading frames predicted to encode homologs of the
Escherichia coli
ferric citrate uptake regulators FecI and FecR. These putative
Bordetella
regulators may mediate heme-responsive positive transcriptional control of the
bhu
genes.
Multiple Domains Are Required for the Toxic Activity of
Pseudomonas aeruginosa
ExoU
Authors: Finck-Barbançon Viviane , Frank Dara W.
Published: July 2002

Expression of ExoU by
Pseudomonas aeruginosa
is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in
trans
. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other
P. aeruginosa
-encoded type III effectors (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.
Evolution of an Autotransporter: Domain Shuffling and Lateral Transfer from Pathogenic
Haemophilus
to
Neisseria
Authors: Davis Jeamelia , Smith Arnold L. , Hughes William R. , Golomb Miriam
Published: July 2002

The genomes of pathogenic
Haemophilus influenzae
strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed
lav
, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with
lav
are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by
H. influenzae, lav
is restricted in distribution to a few pathogenic strains, including
H. influenzae
biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of
lav
is surprisingly similar to that of a gene previously described for
Neisseria meningitidis
. Sequence comparisons suggest that
lav
was transferred relatively recently from
Haemophilus
to
Neisseria
, shortly before the divergence of
N. meningitidis
and
Neisseria gonorrhoeae
. Segments of
lav
predicted to encode passenger and β-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of
lav
. The unusually mobile
lav
locus joins a growing list of genes transferred from
H. influenzae
to
Neisseria
. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.
Isolation and Characterization of Anaerobic Ethylbenzene Dehydrogenase, a Novel Mo-Fe-S Enzyme
Authors: Johnson Hope A. , Pelletier Dale A. , Spormann Alfred M.
Published: July 2002

The first step in anaerobic ethylbenzene mineralization in denitrifying
Azoarcus
sp. strain EB1 is the oxidation of ethylbenzene to (
S
)-(−)-1-phenylethanol. Ethylbenzene dehydrogenase, which catalyzes this reaction, is a unique enzyme in that it mediates the stereoselective hydroxylation of an aromatic hydrocarbon in the absence of molecular oxygen. We purified ethylbenzene dehydrogenase to apparent homogeneity and showed that the enzyme is a heterotrimer (αβγ) with subunit masses of 100 kDa (α), 35 kDa (β), and 25 kDa (γ). Purified ethylbenzene dehydrogenase contains approximately 0.5 mol of molybdenum, 16 mol of iron, and 15 mol of acid-labile sulfur per mol of holoenzyme, as well as a molydopterin cofactor. In addition to ethylbenzene, purified ethylbenzene dehydrogenase was found to oxidize 4-fluoro-ethylbenzene and the nonaromatic hydrocarbons 3-methyl-2-pentene and ethylidenecyclohexane. Sequencing of the encoding genes revealed that
ebdA
encodes the α subunit, a 974-amino-acid polypeptide containing a molybdopterin-binding domain. The
ebdB
gene encodes the β subunit, a 352-amino-acid polypeptide with several 4Fe-4S binding domains. The
ebdC
gene encodes the γ subunit, a 214-amino-acid polypeptide that is a potential membrane anchor subunit. Sequence analysis and biochemical data suggest that ethylbenzene dehydrogenase is a novel member of the dimethyl sulfoxide reductase family of molybdopterin-containing enzymes.
Insertion-Duplication Mutagenesis of
Neisseria
: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island
Authors: Hamilton Holly L. , Schwartz Kevin J. , Dillard Joseph P.
Published: July 2002

We created plasmids for use in insertion-duplication mutagenesis (IDM) of
Neisseria gonorrhoeae
. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene,
ermC
, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved
ermC
, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of
traG
and
traH
mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.
Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels
Authors: Hansen Martin C. , Nielsen Allan K. , Molin Søren , Hammer Karin , Kilstrup Mogens
Published: July 2002

Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium
Lactococcus lactis
. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the
hrcA-grpE-dnaK
operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.
Cohesin-Dockerin Interactions of Cellulosomal Subunits of
Clostridium cellulovorans
Authors: Park Jae-Seon , Matano Yutaka , Doi Roy H.
Published: July 2002

The cellulosome of
Clostridium cellulovorans
consists of three major subunits: CbpA, EngE, and ExgS. The
C. cellulovorans
scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.
The Alternative Sigma Factor SigH Regulates Major Components of Oxidative and Heat Stress Responses inMycobacterium tuberculosis
Authors: Raman Sahadevan , Song Taeksun , Puyang Xiaoling , Bardarov Stoyan , Jacobs William R. , Husson Robert N.
Published: July 2002

Mycobacterium tuberculosisis a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyteMycobacterium smegmatis. In this work we investigated the role ofsigHin theM.tuberculosisresponse to heat and oxidative stress. We determined that aM.tuberculosis sigHmutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genestrxB2/trxCand a gene of unknown function, Rv2466c, is regulated bysigHvia expression from promoters directly recognized by SigH. We also determined that thesigHmutant is more susceptible to heat stress and that inducible expression of the heat shock genesdnaKandclpBis positively regulated bysigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role ofsigHin theM.tuberculosisstress response, we investigated the regulation of the stress-responsive sigma factor genessigEandsigB. We determined that inducible expression ofsigEis regulated bysigHand that basal and inducible expression ofsigBis dependent onsigEandsigH. These data indicate thatsigHplays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important inM.tuberculosispathogenesis.
An Archaeal Photosignal-Transducing Module Mediates Phototaxis in
Escherichia coli
Authors: Jung Kwang-Hwan , Spudich Elena N. , Trivedi Vishwa D. , Spudich John L.
Published: July 2002

Halophilic archaea, such as
Halobacterium salinarum
and
Natronobacterium pharaonis
, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In
N. pharaonis
, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for
H. salinarum
, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from
H. salinarum
and Tar fusion chimera from
Escherichia coli
membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in
H. salinarum
and
E. coli
membranes, respectively, are similar to those of native NpSRII in
N. pharaonis
. These fusion chimeras mediate retinal-dependent phototaxis responses by
Escherichia coli
, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.
Functions of the Mismatch Repair Gene
mutS
from
Acinetobacter
sp. Strain ADP1
Authors: Young David M. , Ornston L. Nicholas
Published: July 2002

The genus
Acinetobacter
encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges.
Acinetobacter
sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned
mutS
from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of
mutS
resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of
mutS
also abolished the marker-specific variations in transforming efficiency seen in
mutS
+
recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual
Acinetobacter
strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the
Acinetobacter
amino acid sequences.
Transcription of the Gene Mediating Methicillin Resistance in
Staphylococcus aureus
(
mecA
) Is Corepressed but Not Coinduced by Cognate
mecA
and β-Lactamase Regulators
Authors: McKinney Tanya K. , Sharma Vijay K. , Craig William A. , Archer Gordon L.
Published: July 2002

Resistance to β-lactam antibiotics in staphylococci is mediated by
mecA
and
blaZ
, genes encoding a penicillin-binding protein (PBP2a) with low β-lactam affinity and β-lactamase, respectively. The
mec
and
bla
regulators,
mecR1-mecI
and
blaR1-blaI
, respectively, encode inducer-repressors with sufficient amino acid homology to suggest that they could coregulate PBP2a production. In order to test this hypothesis, plasmids containing
mec
and
bla
regulatory sequences were introduced into
Staphylococcus aureus
containing a chromosomal
mecA-lacZ
transcriptional fusion. Corepression was confirmed by demonstrating a gene dosage-dependent reduction in β-galactosidase activity by either MecI or BlaI and additive repression when both were present. Both MecI-MecI and BlaI-BlaI homodimer and MecI-BlaI heterodimer interactions were demonstrated in the yeast two-hybrid assay, and purified MecI and BlaI protected the same
mec
promoter-operator sequences. However, MecI was approximately threefold more effective at
mecA-lacZ
transcriptional repression than was BlaI. While MecI and BlaI displayed similar activity as repressors of
mecA
transcription, there was a marked difference between MecR1 and BlaR1 in the rate and specificity of induction. Induction through BlaR1 by a β-lactam was 10-fold greater than through MecR1 at 60 min and was 81% of maximal by 2 h, while induction through MecR1 never exceeded 20% of maximal. Furthermore, complementation studies showed that MecI- or BlaI-mediated
mecA
transcriptional repression could be relieved by induction through homologous but not heterologous sensor-inducer proteins, demonstrating the repressor specificity of induction.
Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from
Pseudaminobacter salicylatoxidans
Authors: Hintner Jan-Peter , Lechner Christa , Riegert Ulrich , Kuhm Andrea Elisabeth , Storm Thomas , Reemtsma Thorsten , Stolz Andreas
Published: July 2002

In cell extracts of
Pseudaminobacter salicylatoxidans
strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and
1
H and
13
C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from
P. salicylatoxidans
also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that
P. salicylatoxidans
synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.
Involvement of
hyp
Gene Products in Maturation of the H
2
-Sensing [NiFe] Hydrogenase of
Ralstonia eutropha
Authors: Buhrke Thorsten , Bleijlevens Boris , Albracht Simon P. J. , Friedrich Bärbel
Published: July 2002

The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H
2
-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of
Ralstonia eutropha
. The RH is a H
2
-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in
hypA
,
hypB
,
hypC
, and
hypX
and was not substantially diminished in
hypD
- and
hypE
-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of
63
Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.
Size Comparisons among Integral Membrane Transport Protein Homologues in
Bacteria
,
Archaea
, and
Eucarya
Authors: Chung Yong Joon , Krueger Christel , Metzgar David , Saier Milton H.
Published: July 2002

Integral membrane proteins from over 20 ubiquitous families of channels, secondary carriers, and primary active transporters were analyzed for average size differences between homologues from the three domains of life:
Bacteria
,
Archaea
, and
Eucarya
. The results showed that while eucaryotic homologues are consistently larger than their bacterial counterparts, archaeal homologues are significantly smaller. These size differences proved to be due primarily to variations in the sizes of hydrophilic domains localized to the N termini, the C termini, or specific loops between transmembrane α-helical spanners, depending on the family. Within the
Eucarya
domain, plant homologues proved to be substantially smaller than their animal and fungal counterparts. By contrast, extracytoplasmic receptors of ABC-type uptake systems in
Archaea
proved to be larger on average than those of their bacterial homologues, while cytoplasmic enzymes from different organisms exhibited little or no significant size differences. These observations presumably reflect evolutionary pressure and molecular mechanisms that must have been operative since these groups of organisms diverged from each other.
Characterization of the Catalytic Activities of the PhoQ Histidine Protein Kinase of
Salmonella enterica
Serovar Typhimurium
Authors: Montagne Martin , Martel Alexandre , Le Moual Hervé
Published: July 2002

Studies of
Escherichia coli
membranes that were highly enriched in the
Salmonella enterica
serovar Typhimurium PhoQ protein showed that the presence of ATP and divalent cations such as Mg
2+
, Mn
2+
, Ca
2+
, or Ba
2+
resulted in PhoQ autophosphorylation. However, when Mg
2+
or Mn
2+
was present at concentrations higher than 0.1 mM, the kinetics of PhoQ autophosphorylation were strongly biphasic, with a rapid autophosphorylation phase followed by a slower dephosphorylation phase. A fusion protein lacking the sensory and transmembrane domains retained the autokinase activity but could not be dephosphosphorylated when Mg
2+
or Mn
2+
was present at high concentrations. The instability of purified [
32
P]phospho-PhoP in the presence of PhoQ-containing membranes indicated that PhoQ also possesses a phosphatase activity. The PhoQ phosphatase activity was stimulated by increasing the Mg
2+
concentration. These data are consistent with a model in which Mg
2+
binding to the sensory domain of PhoQ coordinately regulates autokinase and phosphatase activities.
ς
B
Activity Depends on RsbU in
Staphylococcus aureus
Authors: Giachino P. , Engelmann S. , Bischoff M.
Published: July 2002

Derivatives of the widely used laboratory strain
Staphylococcus aureus
NCTC8325, which are natural
rsbU
mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor ς
B
. The lack of RsbU prevented the heat-dependent production of ς
B
-controlled transcripts and resulted in reduced H
2
O
2
and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth,
rsbU
mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by ς
B
, making it an excellent target for the study of ς
B
activity in
S. aureus
. Reporter gene experiments, using the firefly luciferase gene (
luc
+) fused to the ς
B
-dependent promoter(s) of
asp23
, revealed that ς
B
is almost inactive in 8325 derivatives.
cis
complementation of the 8325 derivative BB255 with the wild-type
rsbU
gene from strain COL produced the
rsbU
+
derivative GP268, a strain possessing a ς
B
activity profile comparable to that of the
rsbU
+
wild-type strain Newman. In GP268, the heat inducibility of ς
B
-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H
2
O
2
were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of ς
B
in
S. aureus
.
The ATP-Dependent Lon Protease ofSalmonella entericaSerovar Typhimurium Regulates Invasion and Expression of Genes Carried onSalmonellaPathogenicity Island 1
Authors: Takaya Akiko , Tomoyasu Toshifumi , Tokumitsu Akane , Morioka Mizue , Yamamoto Tomoko
Published: July 2002

An early step in the pathogenesis ofSalmonella entericaserovar Typhimurium infection is bacterial penetration of the intestinal epithelium. Penetration requires the expression of invasion genes found inSalmonellapathogenicity island 1 (SPI1). These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins. We report here that the expression of invasion genes and the invasive phenotype ofS. entericaserovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis inEscherichia coli. A disrupted mutant oflonwas able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD. Thelonmutant also showed a dramatic enhancement in transcription of the SPI1 geneshilA,invF,sipA, andsipC. The increases ranged from 10-fold to almost 40-fold. It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions. We found that the disruption oflondoes not abolish the repression ofhilAandsipCexpression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals. Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation ofhilAexpression.
Phylogenetic Analysis Reveals Multiple Lateral Transfers of Adenosine-5′-Phosphosulfate Reductase Genes among Sulfate-Reducing Microorganisms
Authors: Friedrich Michael W.
Published: July 2002

Lateral gene transfer affects the evolutionary path of key genes involved in ancient metabolic traits, such as sulfate respiration, even more than previously expected. In this study, the phylogeny of the adenosine-5′-phosphosulfate (APS) reductase was analyzed. APS reductase is a key enzyme in sulfate respiration present in all sulfate-respiring prokaryotes. A newly developed PCR assay was used to amplify and sequence a fragment (∼900 bp) of the APS reductase gene,
apsA
, from a taxonomically wide range of sulfate-reducing prokaryotes (
n
= 60). Comparative phylogenetic analysis of all obtained and available ApsA sequences indicated a high degree of sequence conservation in the region analyzed. However, a comparison of ApsA- and 16S rRNA-based phylogenetic trees revealed topological incongruences affecting seven members of the
Syntrophobacteraceae
and three members of the
Nitrospinaceae
, which were clearly monophyletic with gram-positive sulfate-reducing bacteria (SRB). In addition,
Thermodesulfovibrio islandicus
and
Thermodesulfobacterium thermophilum
,
Thermodesulfobacterium commune
, and
Thermodesulfobacterium hveragerdense
clearly branched off between the radiation of the δ-proteobacterial gram-negative SRB and the gram-positive SRB and not close to the root of the tree as expected from 16S rRNA phylogeny. The most parsimonious explanation for these discrepancies in tree topologies is lateral transfer of
apsA
genes across bacterial divisions. Similar patterns of insertions and deletions in ApsA sequences of donor and recipient lineages provide additional evidence for lateral gene transfer. From a subset of reference strains (
n
= 25), a fragment of the dissimilatory sulfite reductase genes (
dsrAB
), which have recently been proposed to have undergone multiple lateral gene transfers (M. Klein et al., J. Bacteriol. 183:6028–6035, 2001), was also amplified and sequenced. Phylogenetic comparison of DsrAB- and ApsA-based trees suggests a frequent involvement of gram-positive and thermophilic SRB in lateral gene transfer events among SRB.
Energetics of
Helicobacter pylori
and Its Implications for the Mechanism of Urease-Dependent Acid Tolerance at pH 1
Authors: Stingl Kerstin , Uhlemann Eva-Maria , Schmid Roland , Altendorf Karlheinz , Bakker Evert P.
Published: July 2002

In the presence of urea the neutrophilic human pathogen
Helicobacter pylori
survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of
H. pylori
were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea,
H. pylori
accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in
H. pylori
is discussed.
Identification of Two Quorum-Sensing Systems in
Sinorhizobium meliloti
Authors: Marketon Melanie M. , González Juan E.
Published: July 2002

Sinorhizobium meliloti
is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (
Medicago sativa
). This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing. In this study, we characterized the production of
N
-acyl homoserine lactones (AHLs) by two commonly used
S. meliloti
strains, AK631 and Rm1021. We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules. To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis. With this screening method, chromosomal
groEL
(
groELc
) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021. We then used the screening procedure to identify a mutation in a gene homologous to
traM
of
Agrobacterium tumefaciens
, which was able to suppress the phenotype of the
groELc
mutation. A
traR
homolog was identified immediately upstream of
traM
, and we propose that its gene product requires a functional
groELc
for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631. We show that the
traR
/
traM
locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer. We also present evidence for the existence of a second quorum-sensing system,
sinR
/
sinI
, which is present in both AK631 and Rm1021.
Genetic Evidence that the
uvsE
Gene Product of
Deinococcus radiodurans
R1 Is a UV Damage Endonuclease
Authors: Earl Ashlee M. , Rankin Sara K. , Kim Kwang-Pyo , Lamendola Oleana N. , Battista John R.
Published: July 2002

An in vitro transposition system, developed to facilitate gene disruption in
Deinococcus radiodurans
R1, has been used to inactivate the gene designated
dr1819
in

uvrA-1
+

and
uvrA-1
backgrounds.
dr1819
encodes a protein with homology to a UV DNA damage endonuclease expressed by
Schizosaccharomyces pombe
. Interruption of
dr1819
greatly sensitizes the
uvrA-1
strain but not the

uvrA-1
+

strain to UV light, indicating that the
dr1819
gene product is a component in a DNA repair pathway that can compensate for the loss of nucleotide excision repair in this species. Clones of
dr1819
will restore UV resistance to UVS78, a
uvrA-1 uvsE
strain, indicating that
dr1819
and
uvsE
are the same locus.