bluuebunny/crossref_metadata_2025 huggingface dataset:
The Hydrogenase Cytochrome
b
Heme Ligands of
Azotobacter vinelandii
Are Required for Full H
2
Oxidation Capability
Authors: Meek Laura , Arp Daniel J.
Published: July 2002

The hydrogenase in
Azotobacter vinelandii
, like other membrane-bound [NiFe] hydrogenases, consists of a catalytic heterodimer and an integral membrane cytochrome
b
. The histidines ligating the hemes in this cytochrome
b
were identified by H
2
oxidation properties of altered proteins produced by site-directed mutagenesis. Four fully conserved and four partially conserved histidines in HoxZ were substituted with alanine or tyrosine. The roles of these histidines in HoxZ heme binding and hydrogenase were characterized by O
2
-dependent H
2
oxidation and H
2
-dependent methylene blue reduction in vivo. Mutants H33A/Y (H33 replaced by A or Y), H74A/Y, H194A, H208A/Y, and H194,208A lost O
2
-dependent H
2
oxidation activity, H194Y and H136A had partial activity, and H97Y,H98A and H191A had full activity. These results suggest that the fully conserved histidines 33, 74, 194, and 208 are ligands to the hemes, tyrosine can serve as an alternate ligand in position 194, and H136 plays a role in H
2
oxidation. In mutant H194A/Y, imidazole (Imd) rescued H
2
oxidation activity in intact cells, which suggests that Imd acts as an exogenous ligand. The heterodimer activity, quantitatively determined as H
2
-dependent methylene blue reduction, indicated that the heterodimers of all mutants were catalytically active. H33A/Y had wild-type levels of methylene blue reduction, but the other HoxZ ligand mutants had significantly less than wild-type levels. Imd reconstituted full methylene blue reduction activity in mutants H194A/Y and H208A/Y and partial activity in H194,208A. These results indicate that structural and functional integrity of HoxZ is required for physiologically relevant H
2
oxidation, and structural integrity of HoxZ is necessary for full heterodimer-catalyzed H
2
oxidation.
First Evidence for Existence of an Uphill Electron Transfer through the
bc
1
and NADH-Q Oxidoreductase Complexes of the Acidophilic Obligate Chemolithotrophic Ferrous Ion-Oxidizing Bacterium
Thiobacillus ferrooxidans
Authors: Elbehti A. , Brasseur G. , Lemesle-Meunier D.
Published: July 2002

The energy-dependent electron transfer pathway involved in the reduction of pyridine nucleotides which is required for CO
2
fixation to occur in the acidophilic chemolithotrophic organism
Thiobacillus ferrooxidans
was investigated using ferrocytochrome
c
as the electron donor. The experimental results show that this uphill pathway involves a
bc
1
and an NADH-Q oxidoreductase complex functioning in reverse, using an electrochemical proton gradient generated by ATP hydrolysis. Based on these results, a model is presented to explain the balance of the reducing equivalent from ferrocytochrome
c
between the exergonic and endergonic electron transfer pathways.
Saccharomyces cerevisiae
Σ1278b Has Novel Genes of the
N
-Acetyltransferase Gene Superfamily Required for
l
-Proline Analogue Resistance
Authors: Takagi Hiroshi , Shichiri Mika , Takemura Miho , Mohri Miho , Nakamori Shigeru
Published: July 2002

We discovered on the chromosome of
Saccharomyces cerevisiae
Σ1278b novel genes involved in
l
-proline analogue
l
-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the
l
-azetidine-2-carboxylic acid-resistant mutant derived from a cross between
S. cerevisiae
strains S288C and Σ1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for
l
-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the
N
-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for
l
-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Σ1278b background strains. When this novel
MPR1
or
MPR2
gene (sigma 1278b gene for
l
-proline analogue resistance) was introduced into the other
S. cerevisiae
strains, all of the recombinants were resistant to
l
-azetidine-2-carboxylic acid, indicating that both
MPR1
and
MPR2
are expressed and have a global function in
S. cerevisiae
.
ς
BldN
, an Extracytoplasmic Function RNA Polymerase Sigma Factor Required for Aerial Mycelium Formation in
Streptomyces coelicolor
A3(2)
Authors: Bibb Maureen J. , Molle Virginie , Buttner Mark J.
Published: July 2002

Sporulation mutants of
Streptomyces coelicolor
appear white because they are defective in the synthesis of the gray polyketide spore pigment, and such white (
whi
) mutants have been used to define 13 sporulation loci.
whiN
, one of five new
whi
loci identified in a recent screen of NTG (
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine)-induced
whi
strains (N. J. Ryding et al., J. Bacteriol. 181:5419–5425, 1999), was defined by two mutants, R112 and R650. R650 produced frequent spores that were longer than those of the wild type. In contrast, R112 produced long, straight, undifferentiated hyphae, although rare spore chains were observed, sometimes showing highly irregular septum placement. Subcloning and sequencing showed that
whiN
encodes a member of the extracytoplasmic function subfamily of RNA polymerase sigma factors and that the sigma factor has an unusual N-terminal extension of approximately 86 residues that is not present in other sigma factors. A constructed
whiN
null mutant failed to form aerial mycelium (the “bald” phenotype) and, as a consequence,
whiN
was renamed
bldN
. This observation was not totally unexpected because, on some media, the R112 point mutant produced substantially less aerial mycelium than its parent, M145. The
bldN
null mutant did not fit simply into the extracellular signaling cascade proposed for
S. coelicolor bld
mutants. Expression of
bldN
was analyzed during colony development in wild-type and aerial mycelium-deficient
bld
strains.
bldN
was transcribed from a single promoter,
bldNp. bldN
transcription was developmentally regulated, commencing approximately at the time of aerial mycelium formation, and depended on
bldG
and
bldH
, but not on
bldA
,
bldB
,
bldC
,
bldF
,
bldK
, or
bldJ
or on
bldN
itself. Transcription from the
p1
promoter of the response-regulator gene
bldM
depended on
bldN
in vivo, and the
bldMp1
promoter was shown to be a direct biochemical target for ς
BldN
holoenzyme in vitro.
Recovery of Hydrogen Peroxide-Sensitive Culturable Cells of
Vibrio vulnificus
Gives the Appearance of Resuscitation from a Viable but Nonculturable State
Authors: Bogosian Gregg , Aardema Noelle D. , Bourneuf Edward V. , Morris Patricia J. L. , O'Neil Julia P.
Published: July 2002

The viabilities of five strains of
Vibrio vulnificus
were evaluated during the storage of the organisms in sterile seawater at 5°C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to “resuscitate” nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.
Revised Translation Start Site for
secM
Defines an Atypical Signal Peptide That Regulates
Escherichia coli secA
Expression
Authors: Sarker Shameema , Rudd Kenneth E. , Oliver Donald
Published: July 2002

The secretion-responsive regulation of
Escherichia coli secA
occurs by coupling its translation to the translation and secretion of an upstream regulator,
secM
(formerly geneX). We revise the translational start site for
secM
, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for proper
secA
regulation.
Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of
Corynebacterium diphtheriae
That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength
Authors: Lee John H. , Holmes Randall K.
Published: July 2002

The diphtheria toxin repressor (DtxR) of
Corynebacterium diphtheriae
uses Fe
2+
as a corepressor. Holo-DtxR inhibits transcription from the iron-regulated promoters (IRPs) designated IRP1 through IRP5 as well as from the promoters for the
tox
and
hmuO
genes. DtxR binds to 19-bp operators with the consensus sequence 5′-TTAGGTTAGCCTAACCTAA-3′, a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5′-TTAGGT
G
AG
A
C
GC
ACC
C
A
T
-3′ [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5′ end with the putative −10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or −7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(−7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(−7) except for A(−7)C dramatically decreased IRP3 promoter strength. In contrast, the A(−7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5′-TTAGGTTAGCCAAACCTTT-3′) includes the −35 region of the IRP3 promoter. A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and −7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have significant effects on promoter activity for IRP3 and IRP1.
SarA Represses
agr
Operon Expression in a Purified In Vitro
Staphylococcus aureus
Transcription System
Authors: Chakrabarti Swarup K. , Misra Tapan K.
Published: July 2002

Mutation and genetic complementation studies suggested that two chromosomal loci,
agr
and
sar
, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in
Staphylococcus aureus
. We purified recombinant T7-tagged SarA from
Escherichia coli
and determined its effect on transcription from several
S. aureus
promoters by using purified RNA polymerase reconstituted with either ς
A
or ς
B
from
S. aureus
. Of the seven ς
A
-dependent promoters that we tested, SarA repressed transcription from
agr
P2,
agr
P3,
cna
,
sar
P1, and
sea
promoters and did not affect
sec
and
znt
promoters. Furthermore, SarA had no effect on transcription from the ς
B
-dependent
sar
P3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.
Aerobic Activity of
Escherichia coli
Alcohol Dehydrogenase Is Determined by a Single Amino Acid
Authors: Holland-Staley Carol A. , Lee KangSeok , Clark David P. , Cunningham Philip R.
Published: July 2002

Expression of the alcohol dehydrogenase gene,
adhE
, in
Escherichia coli
is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the
adhE
+
structural gene was cloned into expression vectors under the control of the
lacZ
and
trp
c
promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (
adhC
) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the
adhE
structural gene from one of the
adhC
mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the
adhE
gene from the
adhC
mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus,
adhC
strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from
adhC
strains was designated
adhE
*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the
adhE
* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that
adhE
expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in
E. coli
is physiologically significant.
Second-Site Suppressor Mutations of Inactivating Substitutions at Gly247 of the Tetracycline Efflux Protein, Tet(B)
Authors: Saraceni-Richards Cynthia A. , Levy Stuart B.
Published: July 2002

An Asp or Asn substitution for Gly247 in transmembrane helix 8 (TM-8) of Tet(B), the tetracycline efflux protein, eliminated tetracycline resistance. Second site suppressor mutations which partially restored resistance were located in TM-5, -8, -10, or -11 or in cytoplasmic loop 8-9 or loop 10-11. These results indicate physical proximity or functional relationships between TM-8 and these other regions of Tet(B).
Interspecies Complementation of
Escherichia coli ccm
Mutants: CcmE (CycJ) from
Bradyrhizobium japonicum
Acts as a Heme Chaperone during Cytochrome
c
Maturation
Authors: Schulz Henk , Thöny-Meyer Linda
Published: July 2002

Biogenesis of
c
-type cytochromes in α- and γ-proteobacteria requires the function of a set of orthologous genes (
ccm
genes) that encode specific maturation factors. The
Escherichia coli
CcmE protein is a periplasmic heme chaperone. The membrane protein CcmC is required for loading CcmE with heme. By expressing CcmE (CycJ) from
Bradyrhizobium japonicum
in
E. coli
we demonstrated that heme is bound covalently to this protein at a strictly conserved histidine residue. The
B. japonicum
homologue can transfer heme to apocytochrome
c
in
E. coli
, suggesting that it functions as a heme chaperone. CcmC (CycZ) from
B. japonicum
expressed in
E. coli
was capable of inserting heme into CcmE.
TheSaccharomyces cerevisiae ICL2Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism
Authors: Luttik Marijke A. H. , Kötter Peter , Salomons Florian A. , van der Klei Ida J. , van Dijken Johannes P. , Pronk Jack T.
Published: July 2002

TheSaccharomyces cerevisiae ICL1gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologousICL2gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced,ICL2cannot complement the growth defect oficl1null mutants. It has therefore been suggested thatICL2encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whetherICL2encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-typeS. cerevisiaeand of isogenicicl1,icl2, andicl1 icl2null mutants. Isocitrate lyase activity was absent inicl1andicl1 icl2null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and singleiclmutants but not in theicl1 icl2mutant. This demonstrated thatICL2encodes a specific 2-methylisocitrate lyase and that theICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that theICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that ofICL1, transcription ofICL2is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction ofICL2mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.
ModE-Dependent Molybdate Regulation of the Molybdenum Cofactor OperonmoainEscherichia coli
Authors: Anderson Lisa A. , McNairn Elizabeth , Leubke Torben , Pau Richard N. , Boxer David H.
Published: July 2002

The expression of themoalocus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition,moaexpression exhibits a strong requirement for molybdate. The molybdate enhancement ofmoatranscription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of themodoperon encoding the high-affinity molybdate uptake system. Due to the repression ofmoain molybdenum cofactor-sufficient strains, the positive molybdate regulation ofmoais revealed only in strains unable to make the active cofactor. Transcription ofmoais controlled at two sigma-70-type promoters immediately upstream of themoaAgene. Deletion mutations covering the region upstream ofmoaAhave allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction ofmoais exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator ofmoa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affectsmoaexpression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression ofmoa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression ofmoa. The complex control ofmoais discussed in relation to the synthesis of molybdoenzymes in the bacterium.
Construction and Characterization of a
recA
Mutant of
Thiobacillus ferrooxidans
by Marker Exchange Mutagenesis
Authors: Liu Zhenying , Guiliani Nicolas , Appia-Ayme Corinne , Borne Françoise , Ratouchniak Jeanine , Bonnefoy Violaine
Published: July 2002

To construct
Thiobacillus ferrooxidans
mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from
Escherichia coli
to two private
T. ferrooxidans
strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the
T. ferrooxidans recA
gene, a mobilizable suicide plasmid carrying the ATCC 33020
recA
gene disrupted by a kanamycin resistance gene was transferred from
E. coli
to
T. ferrooxidans
ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the
recA
gene, indicating that the
rec
A::Ω-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and γ irradiation compared to the wild-type strain. However, the
T. ferrooxidans recA
mutants are less sensitive to these physical DNA-damaging agents than the
recA
mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in
T. ferrooxidans
.
Genetic Analysis of Functions Involved in Adhesion of
Pseudomonas putida
to Seeds
Authors: Espinosa-Urgel Manuel , Salido Amparo , Ramos Juan-Luis
Published: July 2002

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step.
Pseudomonas putida
KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by
P. putida
KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in
P. putida
, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.
The
chvH
Locus of
Agrobacterium
Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis
Authors: Peng Wen-Tao , Banta Lois M. , Charles Trevor C. , Nester Eugene W.
Published: July 2002

The virulence of
Agrobacterium tumefaciens
depends on both chromosome- and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus,
chvH
, previously identified by Tn
phoA
mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in
Escherichia coli
. The analysis of
vir-lacZ
and
vir-phoA
translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the
chvH
mutant compared with the wild-type strain. The
E. coli efp
gene complemented detergent sensitivity, virulence, and expression of VirE2 in the
chvH
mutant, suggesting that
chvH
and
efp
are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in
A. tumefaciens
. We constructed a
chvH
deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of
Agrobacterium
.
Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of
Staphylococcus aureus
RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase
Authors: Kiriukhin Michael Y. , Debabov Dmitri V. , Shinabarger Dean L. , Neuhaus Francis C.
Published: July 2002

In
Staphylococcus aureus
RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (
ypfP
) which encodes diglucosyldiacylglycerol synthase was recently cloned from
Bacillus subtilis
and expressed in
Escherichia coli
(P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419–430, 1998). To define the role of
ypfP
in this strain of
S. aureus
, a fragment of
ypfP
truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate
ypfP
. Chloramphenicol-resistant (
ypfP
::
cat
) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in
S. aureus
providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either
l
- or
d
-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1%
d
-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the
ypfP
::
cat
mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.
Ralstonia solanacearum
Needs Motility for Invasive Virulence on Tomato
Authors: Tans-Kersten Julie , Huang Huayu , Allen Caitilyn
Published: July 2002

Ralstonia solanacearum
, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that
R. solanacearum
is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the
R. solanacearum
flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (
fliC
), and for the flagellar motor switch protein (
fliM
) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted
R. solanacearum
FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking
fliC
or
fliM
were created by replacing the genes with marked interrupted constructs. Since
fliM
is embedded in the
fliLMNOPQR
operon, the
aphA
cassette was used to make a nonpolar
fliM
mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The
fliC
mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the
fliC
mutant lacked a 30-kDa band corresponding to flagellin. The
fliM
mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the
fliC
mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.
Genome of the Bacterium
Streptococcus pneumoniae
Strain R6
Authors: Hoskins JoAnn , Alborn William E. , Arnold Jeffrey , Blaszczak Larry C. , Burgett Stanley , DeHoff Bradley S. , Estrem Shawn T. , Fritz Lori , Fu Dong-Jing , Fuller Wendy , Geringer Chad , Gilmour Raymond , Glass Jennifer S. , Khoja Hamid , Kraft Angelika R. , Lagace Robert E. , LeBlanc Donald J. , Lee Linda N. , Lefkowitz Elliot J. , Lu Jin , Matsushima Patti , McAhren Scott M. , McHenney Margaret , McLeaster Kevin , Mundy Christopher W. , Nicas Thalia I. , Norris Franklin H. , O'Gara MaryJeanne , Peery Robert B. , Robertson Gregory T. , Rockey Pamela , Sun Pei-Ming , Winkler Malcolm E. , Yang Yong , Young-Bellido Michelle , Zhao Genshi , Zook Christopher A. , Baltz Richard H. , Jaskunas S. Richard , Rosteck Paul R. , Skatrud Paul L. , Glass John I.
Published: July 2002

Streptococcus pneumoniae
is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium
S. pneumoniae
R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.
Improved Soybean Root Association of N-Starved
Bradyrhizobium japonicum
Authors: López-Garcı́a Silvina L. , Vázquez Tirso E. E. , Favelukes Gabriel , Lodeiro Anı́bal R.
Published: July 2002

In this study, we addressed the effects of N limitation in
Bradyrhizobium japonicum
for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day
−1
in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH
4
)
2
SO
4
] limited to only 20 μM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH
4
)
2
SO
4
. The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied
nodC
gene induction in relation to N starvation. In the chromosomal
nodC
::
lacZ
fusion Bj110-573,
nodC
gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures,
nodC
gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.
Identification of Essential Charged Residues in Transmembrane Segments of the Multidrug Transporter MexB of
Pseudomonas aeruginosa
Authors: Guan Lan , Nakae Taiji
Published: July 2002

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on
Pseudomonas aeruginosa
. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408→Glu/Lys939→Arg mutant retained significant activity, while an Asp407→Glu/Lys939→Arg mutant exhibited only marginal function. An Asp407→Glu/Asp408→Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407→Glu/Asp408→Glu/Lys939→Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.
Cloning, Sequences, and Characterization of Two Chitinase Genes from the Antarctic
Arthrobacter
sp. Strain TAD20: Isolation and Partial Characterization of the Enzymes
Authors: Lonhienne Thierry , Mavromatis Konstantinos , Vorgias Constantin E. , Buchon Laurent , Gerday Charles , Bouriotis Vassilis
Published: July 2002

Arthrobacter
sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell.
Arthrobacter
sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (
Ar
ChiA and
Ar
ChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in
Escherichia coli
. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of
Ar
ChiA (881 amino acids [aa]) and
Ar
ChiB (578 aa).
Ar
ChiA and
Ar
ChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of
Ar
ChiA exhibits 55% identity with a chitodextrinase from
Vibrio furnissii
. The
Ar
ChiB catalytic domain exhibits 33% identity with chitinase A of
Bacillus circulans
. The
Ar
ChiA chitin-binding domains are homologous to the chitin-binding domain of
Ar
ChiB.
Ar
ChiA and
Ar
ChiB were purified to homogeneity from the native
Arthrobacter
strain and partially characterized. Thermal unfolding of
Ar
ChiA,
Ar
ChiB, and chitinase A of
Serratia marcescens
was studied using differential scanning calorimetry.
Ar
ChiA and
Ar
ChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.
Programmed Cell Death in
Escherichia coli
: Some Antibiotics Can Trigger
mazEF
Lethality
Authors: Sat Boaz , Hazan Ronen , Fisher Tova , Khaner Hanita , Glaser Gad , Engelberg-Kulka Hanna
Published: July 2002

The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of
Escherichia coli
, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the
E. coli mazEF
system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the
E. coli mazEF
module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.
Functional Characterization of Alanine Racemase from
Schizosaccharomyces pombe
: a Eucaryotic Counterpart to Bacterial Alanine Racemase
Authors: Uo Takuma , Yoshimura Tohru , Tanaka Naotaka , Takegawa Kaoru , Esaki Nobuyoshi
Published: July 2002

Schizosaccharomyces pombe
has an open reading frame, which we named
alr1
+
, encoding a putative protein similar to bacterial alanine racemase. We cloned the
alr1
+
gene in
Escherichia coli
and purified the gene product (Alr1p), with an
M
r
of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent

K
m

and
V
max
values as follows: for
l
-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for
d
-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but
l
-serine and
l
-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of
l
-alanine, respectively.
S. pombe
uses
d
-alanine as a sole nitrogen source, but deletion of the
alr1
+
gene resulted in retarded growth on the same medium. This indicates that
S. pombe
has catabolic pathways for both enantiomers of alanine and that the pathway for
l
-alanine coupled with racemization plays a major role in the catabolism of
d
-alanine.
Saccharomyces cerevisiae
differs markedly from
S. pombe
:
S. cerevisiae
uses
l
-alanine but not
d
-alanine as a sole nitrogen source. Moreover,
d
-alanine is toxic to
S. cerevisiae
. However, heterologous expression of the
alr1
+
gene enabled
S. cerevisiae
to grow efficiently on
d
-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of
d
-alanine.
Cloning and Characterization of the Gene Cluster for Palatinose Metabolism from the Phytopathogenic BacteriumErwinia rhapontici
Authors: Börnke Frederik , Hajirezaei Mohammad , Sonnewald Uwe
Published: July 2002

Erwinia rhaponticiis able to convert sucrose into isomaltulose (palatinose, 6-O-α-d-glucopyranosyl-d-fructose) and trehalulose (1-O-α-d-glucopyranosyl-d-fructose) by the activity of a sucrose isomerase. These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen. This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption. Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated. Here we describe for the first time the cloning and characterization of the palatinose (pal) genes fromErwinia rhapontici. To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned. Thepalgene products ofErwinia rhaponticiwere shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms. ThepalE, palF, palG, palH, palK, palQ, andpalZgenes were oriented divergently with respect to thepalRandpalIgenes, and sequence analysis suggested that the first set of genes constitutes an operon. Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of thepalIgene and thepalEFGHKQZgenes is oppositely regulated at the transcriptional level. Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose. Palatinose activation is inhibited by sucrose. Functional expression ofpalIandpalQinEscherichia colirevealed sucrose isomerase and palatinase activity, respectively.
Functional Analysis of the
Erwinia herbicola tutB
Gene and Its Product
Authors: Katayama Takane , Suzuki Hideyuki , Koyanagi Takashi , Kumagai Hidehiko
Published: July 2002

The
tutB
gene, which lies just downstream of
tpl
, has been cloned from
Erwinia herbicola
, and its product was analyzed. Despite its high sequence similarity to tryptophan transporters, TutB was found to be a tyrosine-specific transporter. Tryptophan acted as a competitive inhibitor of tyrosine transport. Unlike the tryptophanase operon, the
tpl
and
tutB
genes do not constitute an operon.
Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes
Authors: Murata Takahiro , Ohnishi Makoto , Ara Takeshi , Kaneko Jun , Han Chang-Gyun , Li Yong Fang , Takashima Kayoko , Nojima Hideaki , Nakayama Keisuke , Kaji Akira , Kamio Yoshiyuki , Miki Takeyoshi , Mori Hirotada , Ohtsubo Eiichi , Terawaki Yoshiro , Hayashi Tetsuya
Published: July 2002

Rts1, a large conjugative plasmid originally isolated fromProteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence oftus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.
Quorum-Sensing Signals and Quorum-Sensing Genes in
Burkholderia vietnamiensis
Authors: Conway Barbara-Ann , Greenberg E. P.
Published: July 2002

Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many
Proteobacteria
including a clinical isolate of
Burkholderia cepacia
. The
B. cepacia
isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of
Burkholderia vietnamiensis
. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in
B. vietnamiensis
cultures reached concentrations in excess of 20 μM. We isolated a
B. vietnamiensis
DNA fragment containing a gene for the synthesis of decanoyl-HSL (
bviI
) and an open reading frame that codes for a putative signal receptor (
bviR
). A
B. vietnamiensis bviI
mutant did not produce detectable levels of decanoyl-HSL.
Direct Identification of Bacteria from Positive Blood Cultures by Amplification and Sequencing of the 16S rRNA Gene: Evaluation of BACTEC 9240 Instrument True- Positive and False-Positive Results
Authors: Qian Qinfang , Tang Yi-Wei , Kolbert Christopher P. , Torgerson Catherine A. , Hughes John G. , Vetter Emily A. , Harmsen W. Scott , Montgomery Stacy O. , Cockerill Franklin R. , Persing David H.
Published: July 2002

In a previous study which evaluated the BACTEC 9240 automated blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), we noted a 1.3% “instrument false-positive” rate. That is, the BACTEC system signaled that a bottle (BACTEC Plus Aerobic/F bottle or BACTEC Anaerobic Lytic/10 bottle) culture was positive but a Gram stain was negative and there was no growth of bacteria or yeasts on subculture to chocolate agar. Furthermore, from the same sample of blood, cultures for fungi using the Isolator blood culture system (Wampole Laboratories, Cranbury, N.J.) were negative for growth. For the present study, we evaluated 76 instrument false-positive samples for the presence of 16S ribosomal DNA using the MicroSeq 500 kit (PE Biosystems, Foster City, Calif.). These samples also were negative for fungi by the Isolator method. This kit has a PCR module and sequencing module for the amplification and sequencing of the 16S RNA gene and provides a database for sequence alignment and identification of bacteria. To optimize the assay, we evaluated the effect of adding 0.5% bovine serum albumin to the sample from blood culture bottles and found that it decreased the effects of inhibitors on the PCR. Two control groups of blood culture specimens were also evaluated. One group (
n
= 45) were “instrument true positives”; the instrument signaled positive, and subsequent Gram stains were positive and subcultures on chocolate agar grew bacteria. The other group (
n
= 20) were “instrument true negatives”; the instrument signaled negative, the Gram stain was negative, and subcultures on chocolate agar and from the Isolator tube on fungal media showed no growth. None of the 76 instrument false-positive samples had evidence for 16S rRNA gene sequences. All of the instrument true-positive samples and all of the instrument true-negative specimens were positive and negative, respectively, using the MicroSeq 500 kit. Total peripheral white blood cell counts were statistically significantly higher for patients who had instrument false-positive results than for patients who had instrument true-positive or true-negative results (
P
= 0.001). We conclude that instrument false positives signaled by the BACTEC 9240 system are not due to bacteria in the blood culture samples.
Low Concentrations of Mupirocin in the Pharynx following Intranasal Application May Contribute to Mupirocin Resistance in Methicillin-Resistant
Staphylococcus aureus
Authors: Watanabe Hiroshi , Masaki Hironori , Asoh Norichika , Watanabe Kiwao , Oishi Kazunori , Kobayashi Shinobu , Sato Akiyoshi , Sugita Rinya , Nagatake Tsuyoshi
Published: July 2002

We describe a patient with methicillin-resistant
Staphylococcus aureus
(MRSA) colonizing the pharynx. The MIC of mupirocin was 0.25 μg/ml before treatment and increased after treatment to 8 μg/ml. Using pulsed-field gel electrophoresis, we confirmed that the genotypes of MRSA that colonized the pharynx before and after the use of mupirocin were identical. We measured the delivery of mupirocin to the pharynx in three normal volunteers and two patients. Low concentrations of mupirocin were present in the pharynx in all cases 10 min to 3 days after intranasal application. Our data suggested that low concentrations of the drug in the pharynx after intranasal application of mupirocin ointment might explain the selection of mupirocin resistance in MRSA.