bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene Rearrangements in the
vsa
Locus of
Mycoplasma pulmonis
Authors: Shen Xuejun , Gumulak Juliann , Yu Huilan , French C. Todd , Zou Nianxiang , Dybvig Kevin 
Published: July 2002

ABSTRACT

The
vsa
genes of
Mycoplasma pulmonis
encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six
vsa
genes comprising a 10-kb region of the genome of
M. pulmonis
strain KD735-15. In the current study,
vsa
-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11
vsa
genes. The middle region of the
vsa
locus contains a large open reading frame (ORF) that is not a
vsa
gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine
vsa
genes in a strain of
M. pulmonis
that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15
vsaA
, -
C
, -
E
, and -
F
genes and two unique genes (
vsaG
and
vsaH
) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of
vsa
recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the
vrs
box is proposed.
Another Unusual Type of Citric Acid Cycle Enzyme in
Helicobacter pylori
: the Malate:Quinone Oxidoreductase
Authors: Kather Birgit , Stingl Kerstin , van der Rest Michel E. , Altendorf Karlheinz , Molenaar Douwe 
Published: July 2002

ABSTRACT

The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the
Helicobacter pylori
genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of
H. pylori
. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in
mqo
deletion mutants of
Escherichia coli
or
Corynebacterium glutamicum
. Furthermore, this plasmid was able to complement the phenotype of the
C. glutamicum mqo
deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that
H. pylori
possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.
Rho-Dependent Transcription Termination in the
tna
Operon of
Escherichia coli
: Roles of the
boxA
Sequence and the
rut
Site
Authors: Konan Kouacou Vincent , Yanofsky Charles 
Published: July 2002

ABSTRACT

Expression of the tryptophanase (
tna
) operon of
Escherichia coli
is regulated by catabolite repression and by tryptophan-induced transcription antitermination. Tryptophan induction prevents Rho-dependent transcription termination in the leader region of the operon. Induction requires translation of a 24-residue leader peptide-coding region,
tnaC
, containing a single, crucial Trp codon. Studies with a
lacZ
reporter construct lacking the
tnaC-tnaA
spacer region suggest that, in the presence of excess tryptophan, the TnaC leader peptide acts in
cis
on the ribosome translating
tnaC
to inhibit its release. The stalled ribosome is thought to block Rho's access to the transcript. In this paper we examine the roles of the
boxA
sequence and the
rut
site in Rho-dependent termination. Deleting six nucleotides (CGC CCT) of
boxA
or introducing specific point mutations in
boxA
results in high-level constitutive expression. Some constitutive changes introduced in
boxA
do not change the TnaC peptide sequence. We confirm that deletion of the
rut
site results in constitutive expression. We also demonstrate that, in each constitutive construct, replacement of the
tnaC
start codon by a UAG stop codon reduces expression significantly, suggesting that constitutive expression requires translation of the
tnaC
coding sequence. Addition of bicyclomycin, an inhibitor of Rho, to these UAG constructs increases expression, demonstrating that reduced expression is due to Rho action. Combining a
boxA
point mutation with
rut
site deletion results in constitutive expression comparable to that of a maximally induced operon. These results support the hypothesis that in the presence of tryptophan the ribosome translating
tnaC
blocks Rho's access to the
boxA
and
rut
sites, thereby preventing transcription termination.
Two Active Forms of UDP-
N
-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria
Authors: Du Wensheng , Brown James R. , Sylvester Daniel R. , Huang Jianzhong , Chalker Alison F. , So Chi Y. , Holmes David J. , Payne David J. , Wallis Nicola G. 
Published: July 2002

ABSTRACT

Gene sequences encoding the enzymes UDP-
N
-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one
murA
gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two
murA
genes of the gram-positive organism
Streptococcus pneumoniae
were studied further. Each of the
murA
genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its
murA
genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-
N
-acetylglucosamine, confirming they are both active UDP-
N
-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.
Oxazolidinone Resistance Mutations in 23S rRNA of
Escherichia coli
Reveal the Central Region of Domain V as the Primary Site of Drug Action
Authors: Xiong Liqun , Kloss Patricia , Douthwaite Stephen , Andersen Niels Møller , Swaney Steven , Shinabarger Dean L. , Mankin Alexander S. 
Published: July 2002

ABSTRACT

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected
Escherichia coli
oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.
Novel Formaldehyde-Activating Enzyme in
Methylobacterium extorquens
AM1 Required for Growth on Methanol
Authors: Vorholt Julia A. , Marx Christopher J. , Lidstrom Mary E. , Thauer Rudolf K. 
Published: July 2002

ABSTRACT

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the α-proteobacterium
Methylobacterium extorquens
AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from
M. extorquens
AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO
2
. Mutants of
M. extorquens
AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.
Cysteine-Scanning Mutagenesis of the Periplasmic Loop Regions of PomA, a Putative Channel Component of the Sodium-Driven Flagellar Motor in
Vibrio alginolyticus
Authors: Asai Yukako , Shoji Tomokazu , Kawagishi Ikuro , Homma Michio 
Published: July 2002

ABSTRACT

The sodium-driven motor consists of the products of at least four genes,
pomA
,
pomB
,
motX
, and
motY
, in
Vibrio alginolyticus
. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop
1-2
) and at positions 167 to 180 between membrane segments 3 and 4 (loop
3-4
). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop
3-4
impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and
N
-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop
3-4
. None of the loop
1-2
residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.
High-Resolution Physical Map of the
Sinorhizobium meliloti
1021 pSyma Megaplasmid
Authors: Barloy-Hubler Frederique , Capela Delphine , Barnett Melanie J. , Kalman Sue , Federspiel Nancy A. , Long Sharon R. , Galibert Francis 
Published: July 2002

ABSTRACT

To facilitate sequencing of the
Sinorhizobium meliloti
1021 pSyma megaplasmid, a high-resolution map was constructed by ordering 113 overlapping bacterial artificial chromosome clones with 192 markers. The 157 anonymous sequence tagged site markers (81,072 bases) reveal hypothetical functions encoded by the replicon.
Multiple Gene Products and Sequences Required for Excision of the Mobilizable Integrated
Bacteroides
Element NBU1
Authors: Shoemaker Nadja B. , Wang Gui-Rong , Salyers Abigail A. 
Published: July 2002

ABSTRACT

NBU1 is an integrated 10.3-kbp
Bacteroides
element, which can excise and transfer to
Bacteroides
or
Escherichia coli
recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene,
intN1
, was necessary and sufficient for integration of NBU1 into its target site on the
Bacteroides
or
E. coli
genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to
intN1
, four open reading frames plus a large region downstream of the fourth gene,
prmN1
. This downstream sequence was designated XRS, for “excision-required sequence.” XRS contains the
oriT
of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene,
mobN1
. This is the first time an
oriT
, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of
intN1
,
orf2
, completely abolished excision. The next open reading frame,
orf2x
, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of
orf2x
,
orf3
and
prmN1
. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying
prmN1
and part of the XRS (XRS
HIII
) inhibited excision when provided in
trans
on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying
prmN1
, XRS, and the entire mobilization gene,
mobN1
, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a
cis
-acting region (XRS) which includes the
oriT
. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the
oriT
during conjugal transfer, to prevent premature nicking at the
oriT
before NBU1 has excised and circularized.
KorSA from the
Streptomyces
Integrative Element pSAM2 Is a Central Transcriptional Repressor: Target Genes and Binding Sites
Authors: Sezonov Guennadi , Possoz Christophe , Friedmann Annick , Pernodet Jean-Luc , Guérineau Michel 
Published: July 2002

ABSTRACT

pSAM2, a 10.9-kb mobile integrative genetic element from
Streptomyces ambofaciens
, possesses, as do a majority of
Streptomyces
conjugative plasmids, a
kil-kor
system associated with its transfer. The
kor
function of pSAM2 was attributed to the
korSA
gene, but its direct role remained unclear. The present study was focused on the determination of the KorSA targets. It was shown that KorSA acts as a transcriptional repressor by binding to a conserved 17-nucleotide sequence found upstream of only two genes: its own gene,
korSA
, and
pra
, a gene positively controlling pSAM2 replication, integration, and excision. A unique feature of KorSA, compared to Kor proteins from other
Streptomyces
conjugative plasmids, is that it does not directly regulate pSAM2 transfer. KorSA does not bind to the pSAM2 genes coding for transfer and intramycelial spreading. Through the repression of
pra
, KorSA is able to negatively regulate pSAM2 functions activated by Pra and, consequently, to maintain pSAM2 integrated in the chromosome.
Transcriptional and Mutational Analysis of the Uptake Hydrogenase of the Filamentous Cyanobacterium
Anabaena variabilis
ATCC 29413
Authors: Happe Thomas , Schütz Kathrin , Böhme Herbert 
Published: July 2002

ABSTRACT

A 10-kb DNA region of the cyanobacterium
Anabaena variabilis
ATCC 29413 containing the structural genes of the uptake hydrogenase (
hupSL
) was cloned and sequenced. In contrast to the
hupL
gene of
Anabaena
sp. strain PCC 7120, which is interrupted by a 10.5-kb DNA fragment in vegetative cells, there is no programmed rearrangement within the
hupL
gene during the heterocyst differentiation of
A. variabilis
. The
hupSL
genes were transcribed as a 2.7-kb operon and were induced only under nitrogen-fixing conditions, as shown by Northern blot experiments and reverse transcriptase PCR. Primer extension experiments with a fluorescence-labeled oligonucleotide primer confirmed these results and identified the 5′ start of the mRNA transcript 103 bp upstream of the ATG initiation codon. A consensus sequence in the promoter that is recognized by the fumarate nitrate reductase regulator (Fnr) could be detected. The
hupSL
operon in
A. variabilis
was interrupted by an interposon deletion (mutant strain AVM13). Under N
2
-fixing conditions, the mutant strain exhibited significantly increased rates in H
2
accumulation and produced three times more hydrogen than the wild type. These results indicate that the uptake hydrogenase is catalytically active in the wild type and that the enzyme reoxidizes the H
2
developed by the nitrogenase. The Nif phenotype of the mutant strain showed a slight decrease of acetylene reduction compared to that of the wild type.
DnaA, the Initiator of
Escherichia coli
Chromosomal Replication, Is Located at the Cell Membrane
Authors: Newman Gillian , Crooke Elliott 
Published: July 2002

ABSTRACT

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in
Escherichia coli
, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in
E. coli
is a membrane-affiliated event.
Vibrio fischeriGeneshvnAandhvnBEncode Secreted NAD+-Glycohydrolases
Authors: Stabb Eric V. , Reich Karl A. , Ruby Edward G. 
Published: July 2002

ABSTRACTHvnA and HvnB are proteins secreted byVibrio fischeriES114, an extracellular light organ symbiont of the squidEuprymna scolopes, that catalyze the transfer of ADP-ribose from NAD+to polyarginine. Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases (ARTases), and it was hypothesized that they mediate bacterium-host signaling. We have clonedhvnAandhvnBfrom strain ES114.hvnAappears to be expressed as part of a four-gene operon, whereashvnBis monocistronic. The predicted HvnA and HvnB amino acid sequences are 46% identical to one another and share 44% and 34% identity, respectively, with an open reading frame present in thePseudomonas aeruginosagenome. Four lines of evidence indicate that HvnA and HvnB mediate polyarginine ADP-ribosylation not by ARTase activity, but indirectly through an NAD+-glycohydrolase (NADase) activity that releases free, reactive, ADP-ribose: (i) like other NADases, and in contrast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of not only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1,N6-etheno-NAD+and NAD+; (iii) incubation of HvnA and HvnB with [32P]NAD+resulted in the production of ADP-ribose; and (iv) purified HvnA displayed an NADaseVmaxof 400 mol min−1mol−1, which is within the range reported for other NADases and 102- to 104-fold higher than the minor NADase activity reported in bacterial ARTase toxins. Construction and analysis of anhvnA hvnBmutant revealed no other NADase activity in culture supernatants ofV. fischeri, and this mutant initiated the light organ symbiosis and triggered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.
Promoters of the CATG-Specific Methyltransferase Gene
hpyIM
Differ between
iceA1
and
iceA2 Helicobacter pylori
Strains
Authors: Xu Qing , Blaser Martin J. 
Published: July 2002

ABSTRACT

Helicobacter pylori
strains can be divided into two groups, based on the presence of two unrelated genes,
iceA1
and
iceA2,
that occupy the same genomic locus.
hpyIM
, located immediately downstream of either gene, encodes a functional CATG-specific methyltransferase. Despite the strong conservation of the
hpyIM
open reading frame (ORF) among all
H. pylori
strains, the sequences upstream of the ORF in
iceA1
and
iceA2
strains are substantially different. To explore the roles of these upstream sequences in
hpyIM
regulation, promoter analysis of
hpyIM
was performed. Both deletion mutation and primer extension analyses demonstrate that the
hpyIM
promoters differ between
H. pylori
strains 60190 (
iceA1
) and J188 (
iceA2
). In strain 60190,
hpyIM
has two promoters, P
a
or P
I
, which may function independently, whereas only one
hpyIM
promoter, P
c
, was found in strain J188. The XylE assay showed that the
hpyIM
transcription level was much higher in strain 60190 than in strain J188, indicating that regulation of
hpyIM
transcription differs between the
H. pylori iceA1
strain (60190) and
iceA2
strains (J188). Since the
iceA1
and
iceA2
sequences are highly conserved within
iceA1
or
iceA2
strains, we conclude that promoters of the CATG-specific methylase gene
hpyIM
differ between
iceA1
and
iceA2
strains, which leads to differences in regulation of
hpyIM
transcription.