bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium aviumGenes Expressed during Growth in Human Macrophages Detected by Selective Capture of Transcribed Sequences (SCOTS)
Authors: Hou Joan Y. , Graham James E. , Clark-Curtiss Josephine E.
Published: July 2002

Selective capture of transcribed sequences (SCOTS) has been employed to identify 54 cDNA molecules that represent 46 genes that are expressed byMycobacterium aviumduring growth in human macrophages. Some cDNA molecules correspond to genes that are apparently expressed 48 h after infection of macrophages, while others correspond to genes expressed 110 h after infection, and still others correspond to genes expressed throughout the course of infection in our model system. Genes expressed byM. aviumduring growth in macrophages include genes encoding enzymes of several biosynthetic pathways (pyrimidines, mycobactin, and polyketides); genes that encode enzymes involved in intermediary metabolism, energy metabolism (tricarboxylic acid cycle, glyoxalate shunt), and nitrogen metabolism; and genes that encode regulatory proteins. A number of genes of unknown function were also identified, including genes that code for proteins similar to members of the PPE family of proteins ofMycobacterium tuberculosisand proteins similar to those encoded by theM. tuberculosis mcegenes, which have been previously associated with mycobacterial virulence. The SCOTS technique, followed by enrichment for cDNA molecules that are up-regulated or are uniquely expressed byM. aviumduring growth in human macrophages (compared to growth in laboratory broth culture), allows recovery and identification of a greater diversity of cDNA molecules than does subtractive hybridization between cDNA mixtures from macrophage-grown and broth-grownM. avium.Data are presented demonstrating the reproducibility of recovery of a subset of cDNA molecules from cDNA mixtures purified by SCOTS on several different occasions. These results further demonstrate the beneficial utility of the SCOTS technique for identifying genes whose products are needed for successful survival and growth by an organism in a specific environment.
Fibrinogen Binding to Intercellular Adhesion Molecule 1: Implications forPlasmodium falciparumAdhesion
Authors: Gray Carolyn , Craig Alister
Published: July 2002

Intercellular adhesion molecule 1 (ICAM-1) is an endothelial cell adhesion molecule implicated in cerebral malaria. We investigated whether fibrinogen affectsPlasmodium falciparumbinding to ICAM-1, as the ICAM-1 binding sites ofP. falciparumand fibrinogen overlap. We show that fibrinogen dramatically reducesP. falciparumadhesion to ICAM-1 under flow conditions.
ThessuLocus Plays a Key Role in Organosulfur Metabolism inPseudomonas putidaS-313
Authors: Kahnert Antje , Vermeij Paul , Wietek Claudia , James Peter , Leisinger Thomas , Kertesz Michael A.
Published: July 2002

Pseudomonas putidaS-313 can utilize a broad range of aromatic sulfonates as sulfur sources for growth in sulfate-free minimal medium. The sulfonates are cleaved monooxygenolytically to yield the corresponding phenols. miniTn5mutants of strain S-313 which were no longer able to desulfurize arylsulfonates were isolated and were found to carry transposon insertions in thessuEADCBFoperon, which contained genes for an ATP-binding cassette-type transporter (ssuABC), a two-component reduced flavin mononucleotide-dependent monooxygenase (ssuED) closely related to theEscherichia colialkanesulfonatase, and a protein related to clostridial molybdopterin-binding proteins (ssuF). These mutants were also deficient in growth with a variety of other organosulfur sources, including aromatic and aliphatic sulfate esters, methionine, and aliphatic sulfonates other than the natural sulfonates taurine and cysteate. This pleiotropic phenotype was complemented by thessuoperon, confirming its key role in organosulfur metabolism in this species. Further complementation analysis revealed that thessuFgene product was required for growth with all of the tested substrates except methionine and that the oxygenase encoded byssuDwas required for growth with sulfonates or methionine. The flavin reductase SsuE was not required for growth with aliphatic sulfonates or methionine but was needed for growth with arylsulfonates, suggesting that an alternative isozyme exists for the former compounds that is not active in transformation of the latter substrates. Aryl sulfate ester utilization was catalyzed by an arylsulfotransferase, and not by an arylsulfatase as in the related speciesPseudomonas aeruginosa.
lon
Incompatibility Associated with Mutations Causing SOS Induction: Null
uvrD
Alleles Induce an SOS Response in
Escherichia coli
Authors: SaiSree L. , Reddy Manjula , Gowrishankar J.
Published: July 2002

The
uvrD
gene in
Escherichia coli
encodes a 720-amino-acid 3′-5′ DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in
uvrD
are incompatible with
lon
, the incompatibility being a consequence of the chronic induction of SOS in
uvrD
strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease).
uvrD-lon
incompatibility was suppressed by
sulA
,
lexA3
(Ind
−
), or
recA
(Def) mutations. Other mutations, such as
priA
,
dam
,
polA
, and
dnaQ
(
mutD
) mutations, which lead to persistent SOS induction, were also
lon
incompatible. SOS induction was not observed in
uvrC
and
mutH
(or
mutS
) mutants defective, respectively, in excision repair and mismatch repair. Nor was
uvrD
-mediated SOS induction abolished by mutations in genes that affect mismatch repair (
mutH
), excision repair (
uvrC
), or recombination (
recB
and
recF
). These data suggest that SOS induction in
uvrD
mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction in
uvrD
strains.
Phosphorylated PmrA Interacts with the Promoter Region of
ugd
in
Salmonella enterica
Serovar Typhimurium
Authors: Aguirre Andrés , Lejona Sergio , Véscovi Eleonora García , Soncini Fernando C.
Published: July 2002

The
Salmonella
PmrA-PmrB system controls the expression of genes necessary for polymyxin B resistance. Four loci were previously identified as part of the regulon, and interaction of PmrA with the promoter region of three of them was observed. Here we characterized the interaction of PmrA with the promoter region of
ugd
, previously suggested to be regulated indirectly by PmrA. Our results indicate that PmrA controls the expression of
ugd
by interacting with a specific sequence in the promoter region of this gene.
A PhoP-Regulated Outer Membrane Protease of
Salmonella enterica
Serovar Typhimurium Promotes Resistance to Alpha-Helical Antimicrobial Peptides
Authors: Guina Tina , Yi Eugene C. , Wang Houle , Hackett Murray , Miller Samuel I.
Published: July 2002

The outer membrane protein contents of
Salmonella enterica
serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the
Escherichia coli
OmpT and
Yersinia pestis
Pla family of outer membrane proteases.
Salmonella
PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (α-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue α-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to α-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to
Salmonella
resistance to innate immunity.
Cooperative Regulation of
DOG2
, Encoding 2-Deoxyglucose-6-Phosphate Phosphatase, by Snf1 Kinase and the High-Osmolarity Glycerol–Mitogen-Activated Protein Kinase Cascade in Stress Responses of
Saccharomyces cerevisiae
Authors: Tsujimoto Yoshiyuki , Izawa Shingo , Inoue Yoshiharu
Published: July 2002

We screened the genome of
Saccharomyces cerevisiae
for the genes responsive to oxidative stress by using the
lacZ
transposon-insertion library. As a result, we found that expression of the
DOG2
gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of
DOG2
was also induced by osmotic stress. We found a putative
cis
element (STRE, a stress response element) in the
DOG2
promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of
DOG2
gene expression were increased in a
mig1
Δ mutant, while the derepression of
DOG2
was not observed in a
snf1
Δ mutant under glucose-deprived conditions. Induction of the
DOG2
gene expression by osmotic stress was observed in any of the three disruptants
pbs2
Δ,
hog1
Δ, and
snf1
Δ. However, the osmotic induction was completely abolished in both the
snf1Δ pbs2
Δ mutant and the
snf1Δ hog1
Δ mutant. Additionally, these single mutants as well as double mutants failed to induce
DOG2
expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol–mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of
DOG2
.
Energy Taxis Is the Dominant Behavior in
Azospirillum brasilense
Authors: Alexandre Gladys , Greer Suzanne E. , Zhulin Igor B.
Published: July 2002

Energy taxis encompasses aerotaxis, phototaxis, redox taxis, taxis to alternative electron acceptors, and chemotaxis to oxidizable substrates. The signal for this type of behavior is originated within the electron transport system. Energy taxis was demonstrated, as a part of an overall behavior, in several microbial species, but it did not appear as the dominant determinant in any of them. In this study, we show that most behavioral responses proceed through this mechanism in the alpha-proteobacterium
Azospirillum brasilense
. First, chemotaxis to most chemoeffectors typical of the azospirilla habitat was found to be metabolism dependent and required a functional electron transport system. Second, other energy-related responses, such as aerotaxis, redox taxis, and taxis to alternative electron acceptors, were found in
A. brasilense
. Finally, a mutant lacking a cytochrome
c
oxidase of the
cbb
3
type was affected in chemotaxis, redox taxis, and aerotaxis. Altogether, the results indicate that behavioral responses to most stimuli in
A. brasilense
are triggered by changes in the electron transport system.
An
Escherichia coli
Reference Collection Group B2- and Uropathogen-Associated Polymorphism in the
rpoS-mutS
Region of the
E. coli
Chromosome
Authors: Culham Doreen E. , Wood Janet M.
Published: July 2002

Chromosomal DNAs of enterohemorrhagic, uropathogenic, and laboratory attenuated
Escherichia coli
strains differ in the
rpoS-mutS
region. Many uropathogens lack a deletion and an insertion characteristic of enterohemorrhagic strains. At the same chromosomal position, they harbor a 2.1-kb insertion of unknown origin with a base composition suggestive of horizontal gene transfer. Unlike virulence determinants associated with urinary tract infection and/or neonatal meningitis (
pap
or
prs
,
sfa
,
kps
, and
hly
), the 2.1-kb insertion is shared by all group B2 strains of the
E. coli
Reference Collection.
The Histidine Kinase Domain of UhpB Inhibits UhpA Action at the
Escherichia coli uhpT
Promoter
Authors: Wright Jesse S. , Olekhnovich Igor N. , Touchie Gail , Kadner Robert J.
Published: July 2002

The histidine kinase (HK) component of many two-component regulatory systems exhibits regulated ability to phosphorylate itself and to participate in transfer of phosphate to and from its cognate response regulator. The signaling system that controls expression of the UhpT sugar phosphate transporter in
Escherichia coli
in response to external glucose 6-phosphate includes the HK protein UhpB and the polytopic membrane protein UhpC, a UhpT homolog which is required for responsiveness to an inducer and activation of UhpB. The existence of a UhpBC signaling complex is suggested by the requirement for UhpC for the activity of certain constitutively active variants of UhpB, the dominance and epistasis relationships of
uhp
alleles, and the finding that expression of UhpB in excess of UhpC has a strong dominant-negative effect. Expression of a hybrid protein containing the cytoplasmic C-terminal half of UhpB fused to glutathione
S
-transferase (GST) also interfered with Uhp signaling. This interference phenotype could not result solely from the phosphatase activity of UhpB, because interference affected both overexpressed UhpA and UhpA variants which are active in the absence of phosphorylation. Variant forms of UhpB which were active in the absence of UhpC carried amino acid substitutions near motifs conserved in HK proteins. The GST fusion protein inhibited the ability of UhpA to bind and activate transcription at the
uhpT
promoter. Unlike the wild-type situation, a GST fusion variant carrying one of the UhpB-activating substitutions, R324C, displayed autokinase activity and phosphate transfer to UhpA but retained the ability to sequester UhpA when it was altered in the conserved residues important for phosphate transfer. Thus, the default state of UhpB is kinase off, and activation of its phosphate transfer activity requires either the action of UhpC or the occurrence of certain mutations in UhpB. The interference phenotype shown by UhpB in excess of UhpC appears to include the binding and sequestration of UhpA.
Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of
Bacillus subtilis
Authors: Hatt Janet K. , Youngman Philip
Published: July 2002

The Spo0A protein of
Bacillus subtilis
is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-forming
Bacillus
and
Clostridium
and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in
spoIIA
and
spoIIE
promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of
spoIIA
and
spoIIE
, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.
Antigen 43 and Type 1 Fimbriae Determine Colony Morphology of
Escherichia coli
K-12
Authors: Hasman Henrik , Schembri Mark A. , Klemm Per
Published: July 2002

Colony morphology has been used as an important identification and characterization criterion in bacteriology for many decades. However, the molecular mechanisms underlying the appearance of different colony types have been given little attention. The synthesis of O antigen is defunct in
Escherichia coli
K-12, and colonies should accordingly only appear to be rough. However, previous reports have noted the presence of different interchangeable colony morphology types. In this study we have addressed the influence of two phase-variable surface structures, antigen 43 and type 1 fimbriae, on colony morphology. Due to differential expression of these structures, four different colony phenotypes could be distinguished. By creating and studying defined mutants of the respective loci, i.e.,
flu
and
fim
, we conclude that the presence or absence of the corresponding gene products on the cells correlates with the observed colony morphology forms. Interestingly, the habitat specificity of bacteria under static liquid conditions seems to correlate with the colony phenotypes.
Enhanced Nitrogenase Activity in Strains of
Rhodobacter capsulatus
That Overexpress the
rnf
Genes
Authors: Jeong Ho-Sang , Jouanneau Yves
Published: July 2002

In the photosynthetic bacterium
Rhodobacter capsulatus
, a putative membrane-bound complex encoded by the
rnfABCDGEH
operon is thought to be dedicated to electron transport to nitrogenase. In this study, the whole
rnf
operon was cloned under the control of the
nifH
promoter in plasmid pNR117 and expressed in several
rnf
mutants. Complementation analysis demonstrated that transconjugants which integrated plasmid pNR117 directed effective biosynthesis of a functionally competent complex in
R. capsulatus
. Moreover, it was found that strains carrying pNR117 displayed nitrogenase activities 50 to 100% higher than the wild-type level. The results of radioactive labeling experiments indicated that the intracellular content of nitrogenase polypeptides was marginally altered in strains containing pNR117, whereas the levels of the RnfB and RnfC proteins present in the membrane were four- and twofold, respectively, higher than the wild-type level. Hence, the enhancement of in vivo nitrogenase activity was correlated with a commensurate overproduction of the Rnf polypeptides. In vitro nitrogenase assays performed in the presence of an artificial electron donor indicated that the catalytic activity of the enzyme was not increased in strains overproducing the Rnf polypeptides. It is proposed that the supply of reductants through the Rnf complex might be rate limiting for nitrogenase activity in vivo. Immunoprecipitation experiments performed on solubilized membrane proteins revealed that RnfB and RnfC are associated with each other and with additional polypeptides which may be components of the membrane-bound complex.
The RofA Binding Site inStreptococcus pyogenesIs Utilized in Multiple Transcriptional Pathways
Authors: Granok Alexander B. , Parsonage Derek , Ross R. Paul , Caparon Michael G.
Published: July 2002

Understanding the regulation of adhesins defines a pathogenic bacterium's interaction with the local environment within the host. In certain strains ofStreptococcus pyogenes, transcription ofprtF, the gene which encodes the fibronectin-binding adhesin protein F, is activated by RofA under anaerobic conditions. RofA binds specifically to DNA in its target promoters and autoregulates its own expression. In this study, we have used DNase I protection assays to further investigate the interaction of RofA with its target promoters. In the region betweenrofAand the gene which encodes protein F (prtF), RofA binds to two distinct sites: a smaller site (17 bp) adjacent to therofApromoter, and a larger site (40 bp) adjacent to theprtFpromoter. Analysis of fusions to a novel reporter gene whose product consists of the fusion of the N-terminal secretion domain of protein F with the C-terminal enzymatic domain of the enterococcal alkaline phosphatase (PhoZ) revealed that the small RofA binding site had no direct role in control ofprtFtranscription but contributed to regulation ofrofA. Comparison in several strains representing different patterns ofprtFexpression indicated that the larger site was required for activation ofrofAand ofprtFin all strains by both RofA-dependent and -independent pathways. Thus, it would appear that a common recognition sequence provides separate entries to a final common pathway inS. pyogenesvirulence gene expression. The identification of multiple RofA-like proteins and promoters with RofA binding sites implies the existence of a widespread interacting regulatory network.
EFG1
Null Mutants of
Candida albicans
Switch but Cannot Express the Complete Phenotype of White-Phase Budding Cells
Authors: Srikantha Thyagarajan , Tsai Luong K. , Daniels Karla , Soll David R.
Published: July 2002

The
Candida albicans
gene
EFG1
encodes a putative
trans
-acting factor. In strain WO-1, which undergoes the white-opaque transition,
EFG1
is transcribed as a 3.2-kb mRNA in white-phase cells and a less-abundant 2.2-kb mRNA in opaque-phase cells. cDNA sequencing and 5′ rapid amplification of cDNA ends analysis demonstrate that the major difference in molecular mass of the two transcripts is due to different transcription start sites.
EFG1
null mutants form opaque-phase colonies and express the opaque-phase cell phenotype at 25°C. When shifted from 25 to 42°C, mutant opaque-phase cells undergo phenotypic commitment to the white phase, which includes deactivation of the opaque-phase-specific gene
OP4
and activation of the white-phase-specific gene
WH11
, as do wild-type opaque-phase cells. After the commitment event,
EFG1
null mutant cells form daughter cells which have the smooth (pimpleless) surface of white-phase cells but the elongate morphology of opaque-phase cells. Taken together, these results demonstrate that
EFG1
expression is not essential for the switch event per se, but is essential for a subset of phenotypic characteristics necessary for the full expression of the phenotype of white-phase cells. These results demonstrate that
EFG1
is not the site of the switch event, but is, rather, downstream of the switch event.
Replication of Staphylococcal Multiresistance Plasmids
Authors: Firth Neville , Apisiridej Sumalee , Berg Tracey , O'Rourke Brendon A. , Curnock Steve , Dyke Keith G. H. , Skurray Ronald A.
Published: July 2002

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and β-lactamase–heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the
Staphylococcus aureus
plasmids pSK1, pSK41, and pI9789::Tn
552
were found to be related to each other and to the
Staphylococcus xylosus
plasmid pSX267 and are also related to
rep
genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn
552
extended beyond their
rep
genes, encompassing upstream divergently transcribed genes,
orf245
and
orf256
, respectively. Our analyses revealed that genes encoding proteins related to the deduced
orf245
product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that
orf245
is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to
orf245
is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41
rep
gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.
Evidence for the Presence of an F-Type ATP Synthase Involved in Sulfate Respiration in
Desulfovibrio vulgaris
Authors: Ozawa Kiyoshi , Meikari Takanori , Motohashi Ken , Yoshida Masasuke , Akutsu Hideo
Published: July 2002

Using a library of genomic DNA from
Desulfovibrio vulgaris
Miyazaki F, a strict anaerobe, and two synthetic deoxyoligonucleotide probes designed for F-type ATPases, the genes for open reading frames (ORFs) 1 to 5 were cloned and sequenced. The predicted protein sequences of the gene products indicate that they are composed of 172, 488, 294, 471, and 134 amino acids, respectively, and that they share considerable identity at the amino acid level with δ, α, γ, β, and ɛ subunits found in other F-type ATPases, respectively. Furthermore, a component carrying ATPase activity was partially purified from the cytoplasmic membrane fraction of the
D. vulgaris
Miyazaki F cells. The N-terminal amino acid sequences of three major polypeptides separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis were identical to those of the products predicted by the sequences of ORF-2, ORF-3, and ORF-4, suggesting that an F-type ATPase is functioning in the
D. vulgaris
Miyazaki F cytoplasmic membrane. The amount of the F-type ATPase produced in the
D. vulgaris
Miyazaki F cells is similar to that in the
Escherichia coli
cells cultured aerobically. It indicates that the enzyme works as an ATP synthase in the
D. vulgaris
Miyazaki F cells in connection with sulfate respiration.
A
Bacillus subtilis
Gene of Previously Unknown Function,
yhaG
, Is Translationally Regulated by Tryptophan-Activated TRAP and Appears To Be Involved in Tryptophan Transport
Authors: Sarsero Joseph P. , Merino Enrique , Yanofsky Charles
Published: July 2002

Computer analysis of the
Bacillus subtilis
genome sequence revealed a gene with no previously attributed function,
yhaG
, specifying a transcript containing a presumptive binding site for the tryptophan-activated regulatory protein, TRAP. The presumptive TRAP binding site overlaps the
yhaG
Shine-Dalgarno sequence and translation initiation region. TRAP was shown to regulate expression of
yhaG
translationally. Production of the
yhaG
transcript in vivo was found to compete for the binding of TRAP to other known TRAP binding sites. YhaG is likely to be a transmembrane protein involved in tryptophan transport.
Heterologous Expression of Bacterial Epoxyalkane:Coenzyme M Transferase and Inducible Coenzyme M Biosynthesis inXanthobacterStrain Py2 andRhodococcus rhodochrousB276
Authors: Krum Jonathan G. , Ensign Scott A.
Published: July 2002

Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteriaXanthobacterstrain Py2 andRhodococcus rhodochrousstrain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism byR. rhodochrousand demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis.
Transcriptional Organization and Dynamic Expression of the
hbpCAD
Genes, Which Encode the First Three Enzymes for 2-Hydroxybiphenyl Degradation in
Pseudomonas azelaica
HBP1
Authors: Jaspers Marco C. M. , Schmid Andreas , Sturme Mark H. J. , Goslings David A. M. , Kohler Hans-Peter E. , Roelof van der Meer Jan
Published: July 2002

Pseudomonas azelaica
HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes
hbpC
,
hbpA
, and
hbpD
. These three genes form a small noncontiguous cluster. Their expression is activated by the product of regulatory gene
hbpR
, which is located directly upstream of the
hbpCAD
genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators. Transcriptional fusions between the different
hbp
intergenic regions and the
luxAB
genes of
Vibrio harveyi
in
P. azelaica
and in
Escherichia coli
revealed the existence of two HbpR-regulated promoters; one is located in front of
hbpC
, and the other one is located in front of
hbpD
. Northern analysis confirmed that the
hbpC
and
hbpA
genes are cotranscribed, whereas the
hbpD
gene is transcribed separately. No transcripts comprising the entire
hbpCAD
cluster were detected, indicating that transcription from P

hbpC

is terminated after the
hbpA
gene.
E. coli
mutant strains lacking the structural genes for the RNA polymerase ς
54
subunit or for the integration host factor failed to express bioluminescence from P

hbpC

- and P

hbpD

-luxAB
fusions when a functional
hbpR
gene was provided in
trans
. This pointed to the active role of ς
54
and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both P

hbpC

and P

hbpD

contain the typical motifs at position −24 (GG) and −12 (GC) found in ς
54
-dependent promoters. Analysis of changes in the synthesis of the
hbp
mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown
P. azelaica
cells with 2-HBP demonstrated that the specific transcriptional organization of the
hbp
genes ensured smooth pathway expression.
Differential Responses of
Escherichia coli
Cells Expressing Cytoplasmic Domain Mutants of Penicillin-Binding Protein 1b after Impairment of Penicillin-Binding Proteins 1a and 3
Authors: Chalut Christian , Charpentier Xavier , Remy Marie-Hélène , Masson Jean-Michel
Published: July 2002

Penicillin-binding protein 1b (PBP1b) is the major high-molecular-weight PBP in
Escherichia coli
. Although it is coded by a single gene, it is usually found as a mixture of three isoforms which vary with regard to the length of their N-terminal cytoplasmic tail. We show here that although the cytoplasmic tail seems to play no role in the dimerization of PBP1b, as was originally suspected, only the full-length protein is able to protect the cells against lysis when both PBP1a and PBP3 are inhibited by antibiotics. This suggests a specific role for the full-length PBP1b in the multienzyme peptidoglycan-synthesizing complex that cannot be fulfilled by either PBP1a or the shorter PBP1b proteins. Moreover, we have shown by alanine-stretch-scanning mutagenesis that (i) residues R
11
to G
13
are major determinants for correct translocation and folding of PBP1b and that (ii) the specific interactions involving the full-length PBP1b can be ascribed to the first six residues at the N-terminal end of the cytoplasmic domain. These results are discussed in terms of the interactions with other components of the murein-synthesizing complex.
YLR209c
Encodes
Saccharomyces cerevisiae
Purine Nucleoside Phosphorylase
Authors: Lecoq K. , Belloc I. , Desgranges C. , Konrad M. , Daignan-Fornier B.
Published: July 2002

The yeast
YLR209c
(
PNP1
) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of
PNP1
led to inosine and guanosine excretion in the medium, thus showing that
PNP1
plays an important role in the metabolism of these purine nucleosides in vivo.
Transcriptional Regulation of the Two Sterol Esterification Genes in the Yeast
Saccharomyces cerevisiae
Authors: Jensen-Pergakes Kristen , Guo Zhongmin , Giattina Mara , Sturley Stephen L. , Bard Martin
Published: July 2002

Saccharomyces cerevisiae
transcribes two genes,
ARE1
and
ARE2
, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using
ARE1
and
ARE2
promoter fusions to
lacZ
reporters, we demonstrated that transcriptional initiation from the
ARE1
promoter is significantly reduced compared to that from the
ARE2
promoter. Furthermore, the half-life of the
ARE2
mRNA is approximately 12 times as long as that of the
ARE1
transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by
ARE1
is to esterify sterol intermediates, whereas the role of the
ARE2
enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the
ARE1
promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore,
ARE1
and
ARE2
are oppositely regulated by heme. Under heme-deficient growth conditions,
ARE1
was upregulated fivefold while
ARE2
was down-regulated.
ARE2
requires the
HAP1
transcription factor for optimal expression, and both
ARE
genes are derepressed in a
rox1
(repressor of oxygen) mutant genetic background. We further report that the
ARE
genes are not subject to end product inhibition; neither
ARE1
nor
ARE2
transcription is altered in an
are
mutant background, nor does overexpression of either
ARE
gene alter the response of the
ARE-lacZ
reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.
Feedback Regulation of Glucose Transporter Gene Transcription in
Kluyveromyces lactis
by Glucose Uptake
Authors: Milkowski C. , Krampe S. , Weirich J. , Hasse V. , Boles E. , Breunig K. D.
Published: July 2002

In the respirofermentative yeast
Kluyveromyces lactis
, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either
KHT1
and
KHT2
, two tandemly arranged
HXT-
like genes, or
RAG1
, a low-affinity transporter gene that arose by recombination between
KHT1
and
KHT2
. Here we show that
KHT1
is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower
K

m

(3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of
KHT1
and
KHT2
gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a
kht1 kht2
deletion mutant. Thus, the
KHT
genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous β-galactosidase (
LAC4
) promoter and glucose induction of pyruvate decarboxylase were abolished in the
kht1 kht2
mutant. These phenotypes could be partially restored by
HXT
gene family members from
Saccharomyces cerevisiae
. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.
DNA Microarray-Based Identification of Genes Controlled by Autoinducer 2-Stimulated Quorum Sensing in
Escherichia coli
Authors: DeLisa Matthew P. , Wu Chi-Fang , Wang Liang , Valdes James J. , Bentley William E.
Published: July 2002

Bacterial cell-to-cell communication facilitates coordinated expression of specific genes in a growth rate-II and cell density-dependent manner, a process known as quorum sensing. While the discovery of a diffusible
Escherichia coli
signaling pheromone, termed autoinducer 2 (AI-2), has been made along with several quorum sensing genes, the overall number and coordination of genes controlled by quorum sensing through the AI-2 signal has not been studied systematically. We investigated global changes in mRNA abundance elicited by the AI-2 signaling molecule through the use of a
luxS
mutant that was unable to synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the
E. coli
genome, exhibited significant transcriptional changes (either induction or repression) in response to a 300-fold AI-2 signaling differential, with many of the identified genes displaying high induction levels (more than fivefold). Significant induction of
ygeV
, a putative ς
54
-dependent transcriptional activator, and
yhbH
, a ς
54
modulating protein, suggests ς
54
may be involved in
E. coli
quorum sensing.
Biochemical Characterization of Signal Peptidase I from Gram-Positive
Streptococcus pneumoniae
Authors: Peng Sheng-Bin , Wang Li , Moomaw John , Peery Robert B. , Sun Pei-Ming , Johnson Robert B. , Lu Jin , Treadway Patti , Skatrud Paul L. , Wang Q. May
Published: July 2002

Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive
Streptococcus pneumoniae
. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of
S. pneumoniae
signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of
S. pneumoniae
signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases and
Escherichia coli
signal peptidase I. Similar to LexA-like proteases,
S. pneumoniae
signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.
Clock Control of Ultradian Respiratory Oscillation Found during Yeast Continuous Culture
Authors: Murray Douglas B. , Roller Sibel , Kuriyama Hiroshi , Lloyd David
Published: July 2002

A short-period autonomous respiratory ultradian oscillation (period ≈ 40 min) occurs during aerobic
Saccharomyces cerevisiae
continuous culture and is most conveniently studied by monitoring dissolved O
2
concentrations. The resulting data are high quality and reveal fundamental information regarding cellular dynamics. The phase diagram and discrete fast Fourier transformation of the dissolved O
2
values revealed a square waveform with at least eight harmonic peaks. Stepwise changes in temperature revealed that the oscillation was temperature compensated at temperatures ranging from 27 to 34°C when either glucose (temperature quotient [Q
10
] = 1.02) or ethanol (Q
10
= 0.82) was used as a carbon source. After alteration of the temperature beyond the temperature compensation region, phase coherence events for individual cells were quickly lost. As the cell doubling rate decreased from 15.5 to 9.2 h (a factor of 1.68), the periodicity decreased by a factor of 1.26. This indicated that there was a degree of nutrient compensation. Outside the range of dilution rates at which stable oscillation occurred, the mode of oscillation changed. The oscillation in respiratory output is therefore under clock control.
Two-Component Sensor Required for Normal Symbiotic Colonization of
Euprymna scolopes
by
Vibrio fischeri
Authors: Visick Karen L. , Skoufos Line M.
Published: July 2002

The light organ of the squid
Euprymna scolopes
is specifically colonized to a high density by the marine bacterium
Vibrio fischeri
. To date, only a few factors contributing to the specificity of this symbiosis have been identified. Using a genetic screen for random transposon mutants defective in initiating the symbiotic association or in colonizing the light organ to high density, we identified a mutant of
V. fischeri
that exhibited an apparent defect in symbiosis initiation. This mutant was not defective in motility, luminescence, or growth in minimal medium, suggesting that it lacks an essential, previously unidentified symbiotic function. By sequence analysis, we showed that the locus inactivated in this mutant encodes a predicted 927-amino-acid protein with a high degree of similarity to the sensor component of hybrid two-component regulatory systems. We have therefore designated this locus
rscS
, for regulator of symbiotic colonization—sensor. Sequence analysis revealed two hydrophobic regions which may result in the formation of a periplasmic loop involved in signal recognition; PhoA fusion data supported this proposed membrane topology. We have investigated the start site of
rscS
transcription by primer extension and identified a putative promoter region. We hypothesize that RscS recognizes a signal associated with the light organ environment and responds by stimulating a putative response regulator that controls protein function or gene expression to coordinate early colonization events. Further studies on RscS, its cognate response regulator, and the signaling conditions will provide important insight into the interaction between
V. fischeri
and
E. scolopes
.
A Novel Serotype-Specific Gene Cassette (
gltA-gltB
) Is Required for Expression of Teichoic Acid-Associated Surface Antigens in
Listeria monocytogenes
of Serotype 4b
Authors: Lei Xiang-He , Fiedler Franz , Lan Zheng , Kathariou Sophia
Published: July 2002

Listeria monocytogenes
serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes,
gltA
and
gltB
, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either
gltA
or
gltB
. The
gltA
and
gltB
mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within
L. monocytogenes
, only strains of serotypes 4b, 4d, and 4e harbored the
gltA-gltB
cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp
gltA-gltB
cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of
L. monocytogenes
, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.
umuDC
-Mediated Cold Sensitivity Is a Manifestation of Functions of the UmuD
2
C Complex Involved in a DNA Damage Checkpoint Control
Authors: Sutton Mark D. , Walker Graham C.
Published: July 2002

The
umuDC
genes are part of the
Escherichia coli
SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD′
2
C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the
umuDC
gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant
umuC
alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (
umuC104
; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (
umuC125
; A39V), we show that
umuDC
-mediated cold sensitivity can be genetically separated from the role of UmuD′
2
C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that
umuDC
-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that
umuDC
-mediated cold sensitivity is conferred by an activity of the UmuD
2
C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD′ in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the
umuDC
gene products.
Genetic Characterization of a
Sinorhizobium meliloti
Chromosomal Region Involved in Lipopolysaccharide Biosynthesis
Authors: Lagares Antonio , Hozbor Daniela F. , Niehaus Karsten , Otero Augusto J. L. Pich , Lorenzen Jens , Arnold Walter , Pühler Alfred
Published: July 2002

The genetic characterization of a 5.5-kb chromosomal region of
Sinorhizobium meliloti
2011 that contains
lpsB
, a gene required for the normal development of symbiosis with
Medicago
spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes:
greA
and
lpsB
, transcribed in the forward direction; and
lpsE, lpsD, lpsC
, and
lrp
, transcribed in the reverse direction. Except for
lpsB
, none of the
lps
genes were relevant for nodulation and nitrogen fixation. Analysis of the transcriptional organization of
lpsB
showed that
greA
and
lpsB
are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a “nonnitrogen” promoter consensus sequence between
greA
and
lpsB
. The opposite orientation of
lpsB
with respect to its first downstream coding sequence,
lpsE
, indicated that the altered LPS and the defective symbiosis of
lpsB
mutants are both consequences of a primary nonpolar defect in a single gene. Global sequence comparisons revealed that the
greA-lpsB
and
lrp
genes of
S. meliloti
have a genetic organization similar to that of their homologous loci in
R. leguminosarum
bv. viciae. In particular, high sequence similarity was found between the translation product of
lpsB
and a core-related biosynthetic mannosyltransferase of
R. leguminosarum
bv. viciae encoded by the
lpcC
gene. The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the
S. meliloti lpsB
gene restored the wild-type LPS phenotype when introduced into
lpcC
mutants of
R. leguminosarum
. These results support the view that
S. meliloti lpsB
also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of other
lpsB
-homologous sequences in several members of the family
Rhizobiaceae
.