bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemotaxis in
Borrelia burgdorferi
Authors: Shi Wenyuan , Yang Zhaomin , Geng Yongzhi , Wolinsky Lawrence E. , Lovett Michael A.
Published: December 2019

Borrelia burgdorferi
is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of
B. burgdorferi
required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of
B. burgdorferi
. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of
B. burgdorferi
. Rabbit serum was found to be an attractant for
B. burgdorferi
, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g.,
Spirochaeta aurantia
),
B. burgdorferi
did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of
B. burgdorferi.
Direct Sulfhydrylation for Methionine Biosynthesis in
Leptospira meyeri
Authors: Belfaiza J. , Martel A. , Margarita D. , Saint Girons I.
Published: December 2019

A gene library of the
Leptospira meyeri
serovar semaranga strain Veldrat S.173 DNA has been constructed in a mobilizable cosmid with inserts of up to 40 kb. It was demonstrated that a
Leptospira
DNA fragment carrying
metY
complemented
Escherichia coli
strains carrying mutations in
metB
. The latter gene encodes cystathionine γ-synthase, an enzyme which catalyzes the second step of the methionine biosynthetic pathway. The
metY
gene is 1,304 bp long and encodes a 443-amino-acid protein with a molecular mass of 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the
Leptospira metY
product has a high degree of similarity to those of
O
-acetylhomoserine sulfhydrylases from
Aspergillus nidulans
and
Saccharomyces cerevisiae
. A lower degree of sequence similarity was also found with bacterial cystathionine γ-synthase. The
L. meyeri metY
gene was overexpressed under the control of the T7 promoter. MetY exhibits an
O
-acetylhomoserine sulfhydrylase activity. Genetic, enzymatic, and physiological studies reveal that the transsulfuration pathway via cystathionine does not exist in
L. meyeri
, in contrast to the situation found for fungi and some bacteria. Our results indicate, therefore, that the
L. meyeri
MetY enzyme is able to perform direct sulfhydrylation for methionine biosynthesis by using
O
-acetylhomoserine as a substrate.
Reverse Gyrase from the Hyperthermophilic Bacterium
Thermotoga maritima
: Properties and Gene Structure
Authors: de la Tour Claire Bouthier , Portemer Christiane , Kaltoum Habib , Duguet Michel
Published: December 2019

The hyperthermophilic bacterium
Thermotoga maritima
MSB8 possesses a reverse gyrase whose enzymatic properties are very similar to those of archaeal reverse gyrases. It catalyzes the positive supercoiling of the DNA in an Mg
2+
- and ATP-dependent process. Its optimal temperature of activity is around 90°C, and it is highly thermostable. We have cloned and DNA sequenced the corresponding gene (
T. maritima topR
). This is the first report describing the analysis of a gene encoding a reverse gyrase in bacteria. The
T. maritima topR
gene codes for a protein of 1,104 amino acids with a deduced molecular weight of 128,259, a value in agreement with that estimated from the denaturing gel electrophoresis of the purified enzyme. Like its archaeal homologs, the
T. maritima
reverse gyrase exhibits helicase and topoisomerase domains, and its sequence matches very well the consensus sequence for six reverse gyrases now available. Phylogenetic analysis shows that all reverse gyrases, including the
T. maritima
enzyme, form a very homogeneous group, distinct from the type I 5′ topoisomerases of the TopA subfamily, for which we have previously isolated a representative gene in
T. maritima
(
topA
). The coexistence of these two distinct genes, coding for a reverse gyrase and an ω-like topoisomerase, respectively, together with the recent description of a gyrase in
T. maritima
(O. Guipaud, E. Marguet, K. M. Noll, C. Bouthier de la Tour, and P. Forterre, Proc. Natl. Acad. Sci. USA 94:10606–10611, 1977) addresses the question of the control of the supercoiling in this organism.
Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of
Pseudomonas putida
GJ31
Authors: Kaschabek Stefan R. , Kasberg Thomas , Müller Dagmar , Mars Astrid E. , Janssen Dick B. , Reineke Walter
Published: December 2019

A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of
Pseudomonas putida
GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native
M
r
value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate.
YopD of
Yersinia pestis
Plays a Role in Negative Regulation of the Low-Calcium Response in Addition to Its Role in Translocation of Yops
Authors: Williams Andrew W. , Straley Susan C.
Published: December 2019

Yersinia pestis
produces a set of virulence proteins (Yops and LcrV) that are expressed at high levels and secreted by a type III secretion system (Ysc) upon bacterium-host cell contact, and four of the Yops are vectorially translocated into eukaryotic cells. YopD, YopB, and YopK are required for the translocation process. In vitro, induction and secretion occur at 37°C in the absence of calcium. LcrH (also called SycD), a protein required for the stability and secretion of YopD, had initially been identified as a negative regulator of Yop expression. In this study, we constructed a
yopD
mutation in both wild-type and secretion-defective (
ysc
)
Y. pestis
to determine if the
lcrH
phenotype could be attributed to the decreased stability of YopD. These mutants were constitutively induced for expression of Yops and LcrV, despite the presence of the secreted negative regulator LcrQ, demonstrating that YopD is involved in negative regulation, regardless of a functioning Ysc system. Normally, secretion of Yops and LcrV is blocked in the presence of calcium. The single
yopD
mutant was not completely effective in blocking secretion: LcrV was secreted equally well in the presence and absence of calcium, while there was partial secretion of Yops in the presence of calcium. YopD is probably not rate limiting for negative regulation, as increasing levels of YopD did not result in decreased Yop expression. Overexpression of LcrQ in the
yopD
mutant had no significant effect on Yop expression, whereas increased levels of LcrQ in the parent resulted in decreased levels of Yops. These results indicate that LcrQ requires YopD to function as a negative regulator.
Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring
Authors: Hugenholtz Philip , Pitulle Christian , Hershberger Karen L. , Pace Norman R.
Published: December 2019

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609–1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or
Bacteria
-specific rDNA primers and cloned. Unique rDNA types among >300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (≥98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing
Calderobacterium
and the sulfate reducers
Thermodesulfovibrio
and
Thermodesulfobacterium
, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing
Aquificales
and the sulfate-reducing δ-
Proteobacteria
. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that
Archaea
dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.
Staphylococcus aureus cap5O
and
cap5P
Genes Functionally Complement Mutations Affecting Enterobacterial Common-Antigen Biosynthesis in
Escherichia coli
Authors: Kiser Kevin B. , Lee Jean C.
Published: December 2019

The
Staphylococcus aureus cap5P
and
cap5O
genes of the type 5 capsule biosynthetic locus restore enterobacterial common-antigen expression to
Escherichia coli
mutants defective in
rffE
and
rffD
gene expression, respectively. Cap5P and Cap5O likely function as UDP-GlcNAc 2-epimerase and UDP-ManNAc dehydrogenase enzymes, respectively, in the synthesis of the capsule precursor UDP-ManNAcA.
The ATP Synthase
atpHAGDC
(F
1
) Operon from
Rhodobacter capsulatus
Authors: Borghese Roberto , Crimi Massimo , Fava Luca , Melandri Bruno Andrea
Published: December 2019

The
atpHAGDC
operon of
Rhodobacter capsulatus
, containing the five genes coding for the F
1
sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying
atp
deletions in the
R. capsulatus
chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.
Conversion of NfsA, the Major
Escherichia coli
Nitroreductase, to a Flavin Reductase with an Activity Similar to That of Frp, a Flavin Reductase in
Vibrio harveyi
, by a Single Amino Acid Substitution
Authors: Zenno Shuhei , Kobori Toshiro , Tanokura Masaru , Saigo Kaoru
Published: December 2019

NfsA is the major oxygen-insensitive nitroreductase of
Escherichia coli
, similar in amino acid sequence to Frp, a flavin reductase of
Vibrio harveyi
. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.
Domain Structure of the ATP-Binding-Cassette Protein MalK of
Salmonella typhimurium
as Assessed by Coexpressed Half Molecules and LacK′-′MalK Chimeras
Authors: Schmees Günter , Schneider Erwin
Published: December 2019

ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of
Salmonella typhimurium
and
Escherichia coli
is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of
S. typhimurium
. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a
malK
strain and displayed substantial regulatory activity. The same regulatory activity was obtained when
malKC1
was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.
Gram-Negative Bacteria Produce Membrane Vesicles Which Are Capable of Killing Other Bacteria
Authors: Li Zusheng , Clarke Anthony J. , Beveridge Terry J.
Published: December 2019

Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (
Citrobacter
,
Enterobacter
,
Escherichia
,
Klebsiella
,
Morganella
,
Proteus
,
Salmonella
, and
Shigella
strains), lysed many gram-positive (including
Mycobacterium
) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767–2774, 1996). MV-sensitive bacteria possessed A1α, A4α, A1γ, A2α, and A4γ peptidoglycan chemotypes, whereas A3α, A3β, A3γ, A4β, B1α, and B1β chemotypes were not affected.
Pseudomonas aeruginosa
PAO1 vesicles possessed the most lytic activity.
The Nine Genes of the
Nocardia lactamdurans
Cephamycin Cluster Are Transcribed into Large mRNAs from Three Promoters, Two of Them Located in a Bidirectional Promoter Region
Authors: Enguita Francisco J. , Coque Juan Jose R. , Liras Paloma , Martin Juan F.
Published: December 2019

The nine biosynthesis genes of the
Nocardia lactamdurans
cephamycin cluster are expressed as three different mRNAs initiating at promoters
latp
,
cefDp
, and
pcbABp
, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (
latp
and
cefDp
) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (
Streptomyces
antibiotic regulatory proteins) family. The
lat
gene is transcribed in a single monocistronic transcript initiating at
latp
. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (
pcbAB
,
pcbC
,
cmcI
,
cmcJ
,
cefF
, and
cmcH
) started at
pcbABp
. A third polycistronic mRNA corresponding to the
cefD
and
cefE
genes started at
cefDp
.
Identification and Recombinant Expression of a
Mycobacterium avium
Rhamnosyltransferase Gene (
rtfA
) Involved in Glycopeptidolipid Biosynthesis
Authors: Eckstein Torsten M. , Silbaq Fauzi S. , Chatterjee Delphi , Kelly Nathan J. , Brennan Patrick J. , Belisle John T.
Published: December 2019

The
Mycobacterium avium
complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the
M. avium
complex. We previously described the
ser2
gene cluster, encoding the synthesis of the haptenic oligosaccharide (2,3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (
ser2A
) encoding a putative rhamnosyltransferase. Sequencing of the
ser2A
locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in
Mycobacterium smegmatis
, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed
rtfA
, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of
rtfA
will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple
M. avium
complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.
Endochitinase Is Transported to the Extracellular Milieu by theeps-Encoded General Secretory Pathway ofVibrio cholerae
Authors: Connell Terry D. , Metzger Daniel J. , Lynch Jennifer , Folster Jason P.
Published: December 2019

ThechiAgene ofVibrio choleraeencodes a polypeptide which degrades chitin, a homopolymer ofN-acetylglucosamine (GlcNAc) found in cell walls of fungi and in the integuments of insects and crustaceans.chiAhas a coding capacity corresponding to a polypeptide of 846 amino acids having a predicted molecular mass of 88.7 kDa. A 52-bp region with promoter activity was found immediately upstream of thechiAopen reading frame. Insertional inactivation of the chromosomal copy of the gene confirmed that expression of chitinase activity byV. choleraerequiredchiA. Fluorescent analogues were used to demonstrate that the enzymatic activity of ChiA was specific for β,1-4 glycosidic bonds located between GlcNAc monomers in chitin. Antibodies against ChiA were obtained by immunization of a rabbit with a MalE-ChiA hybrid protein. Polypeptides with antigenic similarity to ChiA were expressed by classical and El Tor biotypes ofV. choleraeand by the closely related bacteriumAeromonas hydrophila. Immunoblotting experiments using the wild-type strain 569B and the secretion mutant M14 confirmed that ChiA is an extracellular protein which is secreted by theepssystem. Theepssystem is also responsible for secreting cholera toxin, an oligomeric protein with no amino acid homology to ChiA. These results indicate that ChiA and cholera toxin have functionally similar extracellular transport signals that are essential foreps-dependent secretion.
Multiple In Vivo Roles for the −12-Region Elements of Sigma 54 Promoters
Authors: Wang Lei , Gralla Jay D.
Published: December 2019

Alignment of sigma 54-dependent promoters indicates conservation of two sequence elements. Six nucleotides in the downstream −12 element were mutated individually to each nonconsensus nucleotide. mRNA levels were measured in vivo for each promoter under strongly activating conditions. The results showed that the consensus sequence was not the strongest promoter. Instead, the −12 consensus element consists of two subregions that behave differently when mutated. Single changes in the upstream TTT consensus subregion can lead to increases in transcription, whereas single changes in the downstream GC(A/T) can lead to decreases in transcription. Selected double mutations with changes in both subregions were constructed and studied in vivo. No double mutation increased promoter strength, and some decreased it. Mutant promoters were also assayed under nonactivating conditions in vivo. No mRNA was detected in 23 of the 24 promoters tested. However, one double mutant showed substantial levels of transcript, indicating that the −12 sequence was capable of specifying basal transcription under nonactivating conditions. Overall, the results show that the −12 region has multiple roles in transcription in vivo, including modulating both basal and induced RNA levels.
Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated
Escherichia coli
: Requirement for DNA End Processing
Authors: Thoms Brigitte , Wackernagel Wilfried
Published: December 2019

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m
2
). The activity decrease was seen by an increase in survival of phage T4
2
−
of about 200-fold (phage T4
2
−
has defective duplex DNA end-protecting gene
2
protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with
ssb
+
) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in
xonA
,
recJ
, and
sbcCD
, particularly in the
recJ sbcCD
and
xonA recJ sbcCD
combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.
Regulation of Motility Behavior in
Myxococcus xanthus
May Require an Extracytoplasmic-Function Sigma Factor
Authors: Ward Mandy J. , Lew Helen , Treuner-Lange Anke , Zusman David R.
Published: December 2019

Using interaction trap technology, we identified a putative extracytoplasmic-function (ECF) sigma factor (RpoE1) in
Myxococcus xanthus
, a bacterium which has a complex life cycle that includes fruiting body formation. The first domain of the response regulator protein FrzZ, a component of the Frz signal transduction system, was used as bait. Although the RpoE1 protein displayed no interactions with control proteins presented as bait, a weak interaction with a second
M. xanthus
response regulator (AsgA) was observed. While the specificity of the FrzZ-RpoE1 interaction therefore remains speculative, cloning and sequencing of the region surrounding
rpoE1
localized it to a position downstream of the
frzZ
gene. A potential promoter site for binding of an ECF sigma factor was identified upstream of
rpoE1
, suggesting the gene may be autoregulated. However, primer extension studies suggested that transcription of
rpoE1
occurs under both vegetative and developmental conditions from a ς
70
-like promoter. Dot blot analysis of RNA preparations confirmed the low-level, constitutive expression of
rpoE1
during both stages of the life cycle. Analysis of an insertion mutant also indicated a role for RpoE1 under both vegetative and developmental conditions, since swarming was reduced on nutrient-rich agar and developmental aggregation was effected under starvation conditions, especially at high cell densities. An insertion mutation introduced into the gene directly downstream of
rpoE1
(
orf5
) did not result in either swarming or developmental aggregation defects, even though the gene is transcribed as part of the same operon. Therefore, we propose that this new ECF sigma factor could play a role in the transcriptional regulation of genes involved in motility behavior during both stages of the complex
M. xanthus
life cycle.
Role of the
Escherichia coli
SurA Protein in Stationary-Phase Survival
Authors: Lazar Sara W. , Almirón Marta , Tormo Antonio , Kolter Roberto
Published: December 2019

SurA is a periplasmic peptidyl-prolyl isomerase required for the efficient folding of extracytoplasmic proteins. Although the
surA
gene had been identified in a screen for mutants that failed to survive in stationary phase, the role played by SurA in stationary-phase survival remained unknown. The results presented here demonstrate that the survival defect of
surA
mutants is due to their inability to grow at elevated pH in the absence of ς
S
. When cultures of
Escherichia coli
were grown in peptide-rich Luria-Bertani medium, the majority of the cells lost viability during the first two to three days of incubation in stationary phase as the pH rose to pH 9. At this time the surviving cells resumed growth. In cultures of
surA rpoS
double mutants the survivors lysed as they attempted to resume growth at the elevated pH. Cells lacking penicillin binding protein 3 and ς
S
had a survival defect similar to that of
surA rpoS
double mutants, suggesting that SurA foldase activity is important for the proper assembly of the cell wall-synthesizing apparatus.
Examination of the Role of DNA Polymerase Proofreading in the Mutator Effect of Miscoding tRNAs
Authors: Slupska Malgorzata M. , King Angela G. , Lu Louise I. , Lin Rose H. , Mao Emily F. , Lackey Chantal A. , Chiang Ju-Huei , Baikalov Claudia , Miller Jeffrey H.
Published: December 2019

We previously described
Escherichia coli
mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, ɛ, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered ɛ subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of
mutD
alleles. In the present work, we extended the study of specific
mutD
alleles and constructed 16 altered
mutD
genes by replacing each aspartic acid codon, in series, with a glycine codon in the
dnaQ
gene that encodes ɛ. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the
mutD
gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the ɛ subunit playing a major role in the mutator effect of misreading tRNAs.
CHR, a Novel Family of Prokaryotic Proton Motive Force-Driven Transporters Probably Containing Chromate/Sulfate Antiporters
Authors: Nies Dietrich H. , Koch Sandra , Wachi Shinichiro , Peitzsch Nicola , Saier Milton H.
Published: December 2019

We describe a small family of proteins, CHR, which contains members that function in chromate and/or sulfate transport. CHR proteins occur in bacteria and archaea. They consist of about 400 amino acyl residues, appear to have 10 transmembrane α-helical segments in an unusual 4+6 arrangement, and arose by an intragenic duplication event.
Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase from
Mycobacterium smegmatis
Authors: Boshoff Helena I. M. , Mizrahi Valerie
Published: December 2019

The pyrazinamidase from
Mycobacterium smegmatis
was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of
M. smegmatis
. An open reading frame, designated
pzaA
, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the
pzaA
gene by homologous recombination yielded a mutant,
pzaA
::
aph
, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of
M. smegmatis
. Recombinant forms of the
M. smegmatis
PzaA and the
Mycobacterium tuberculosis
pyrazinamidase/nicotinamidase (PncA) were produced in
Escherichia coli
and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable

K
m

values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for
M. tuberculosis
was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of
pzaA
conferred PZA susceptibility on
M. smegmatis
by reducing the MIC of this drug to 150 μg/ml.
Identification and Characterization of the
fis
Operon in Enteric Bacteria
Authors: Beach Michael B. , Osuna Robert
Published: December 2019

The small DNA binding protein Fis is involved in several different biological processes in
Escherichia coli
. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at
oriC. fis
has also been isolated from
Salmonella typhimurium
, and the genomic sequence of
Haemophilus influenzae
reveals its presence in this bacteria. This work extends the characterization of
fis
to other organisms. Very similar
fis
operon structures were identified in the enteric bacteria
Klebsiella pneumoniae
,
Serratia marcescens
,
Erwinia carotovora
, and
Proteus vulgaris
but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in
K. pneumoniae
,
S. marcescens
,
E. coli
, and
S. typhimurium
and 96 to 98% identical when
E. carotovora
and
P. vulgaris
Fis are considered. The deduced amino acid sequence for
H. influenzae
Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the
E. carotovora
,
P. vulgaris
, and
H. influenzae
Fis proteins are not functionally identical. An open reading frame (ORF1) preceding
fis
in
E. coli
is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the
E. coli fis
P region from −53 to +27 and the region around −116 containing an
ihf
binding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.
Three Replicons of
Rhizobium
sp. Strain NGR234 Harbor Symbiotic Gene Sequences
Authors: Flores Margarita , Mavingui Patrick , Girard Lourdes , Perret Xavier , Broughton William J. , Martínez-Romero Esperanza , Dávila Guillermo , Palacios Rafael
Published: December 2019

Rhizobium
sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234
a
, a megaplasmid (pNGR234
b
), and the chromosome. Symbiotic gene sequences not present in pNGR234
a
were analyzed by hybridization. DNA sequences homologous to the genes
fixLJKNOPQGHIS
were found on the chromosome, while sequences homologous to
nodPQ
and
exoBDFLK
were found on pNGR234
b
.
Complex Metabolic Phenotypes Caused by a Mutation in
yjgF
, Encoding a Member of the Highly Conserved YER057c/YjgF Family of Proteins
Authors: Enos-Berlage Jodi L. , Langendorf Mark J. , Downs Diana M.
Published: December 2019

The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in
Salmonella typhimurium
. Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated. Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis. Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in
yjgF
, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein. The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans. This work represents the first detailed phenotypic characterization of
yjgF
mutants in any organism and provides important clues as to the function of this highly conserved class of proteins. Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.
The First Gene of theBacillus subtilis clpCOperon,ctsR, Encodes a Negative Regulator of Its Own Operon and Other Class III Heat Shock Genes
Authors: Krüger Elke , Hecker Michael
Published: December 2019

TheBacillus subtilis clpCoperon is regulated by two stress induction pathways relying on either ςBor a class III stress induction mechanism acting at a ςA-like promoter. When theclpCoperon was placed under the control of the isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Pspacpromoter, dramatic repression of the naturalclpCpromoters fused to alacZreporter gene was noticed after IPTG induction. This result strongly indicated negative regulation of theclpCoperon by one of its gene products. Indeed, the negative regulator could be identified which is encoded by the first gene of theclpCoperon,ctsR, containing a predicted helix-turn-helix DNA-binding motif. Deletion ofctsRabolished the negative regulation and resulted in high expression of both theclpCoperon and theclpPgene under nonstressed conditions. Nevertheless, a further increase inclpCandclpPmRNA levels was observed after heat shock, even in the absence of ςB, suggesting a second induction mechanism at the vegetative promoter. Two-dimensional gel analysis and mRNA studies showed that the expression of other class III stress genes was at least partially influenced by thectsRdeletion. Studies with differentclpCpromoter fragments either fused to the reporter genebgaBor used in gel mobility shift experiments with the purified CtsR protein revealed a possible target region where the repressor seemed to bind in vivo and in vitro. Our data demonstrate that the CtsR protein acts as a global repressor of theclpCoperon, as well as other class III heat shock genes, by preventing unstressed transcription from either the ςB- or ςA-dependent promoter and might be inactivated or dissociate under inducing stress conditions.
Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria
Authors: Thulasiraman Padmamalini , Newton Salete M. C. , Xu Jide , Raymond Kenneth N. , Mai Christine , Hall Angela , Montague Marjorie A. , Klebba Phillip E.
Published: December 2019

The ligand-gated outer membrane porin FepA serves
Escherichia coli
as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their
59
Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (

K
d

≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K
3
MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from
Bordetella bronchisepticus
,
Pseudomonas aeruginosa
, and
Salmonella typhimurium
in the native organisms and as plasmid-mediated clones expressed in
E. coli
. All the transport proteins bound ferric enterobactin with high affinity (

K
d

≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10
9
cells) in their own particular membrane environments. However, the FepA and IroN proteins of
S. typhimurium
failed to efficiently function in
E. coli
. For
E. coli
,
S. typhimurium
, and
P. aeruginosa
, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.
Sucrose-Phosphate Synthase from
Synechocystis
sp. Strain PCC 6803: Identification of the
spsA
Gene and Characterization of the Enzyme Expressed in
Escherichia coli
Authors: Curatti Leonardo , Folco Eduardo , Desplats Paula , Abratti Gustavo , Limones Verónica , Herrera-Estrella Luis , Salerno Graciela
Published: December 2019

The first identification and characterization of a prokaryotic gene (
spsA
) encoding sucrose-phosphate synthase (SPS) is reported for
Synechocystis
sp. strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium. Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.
The Fur-Regulated Gene Encoding the Alternative Sigma Factor PvdS Is Required for Iron-Dependent Expression of the LysR-Type Regulator PtxR in
Pseudomonas aeruginosa
Authors: Vasil Michael L. , Ochsner Urs A. , Johnson Zaiga , Colmer Jane A. , Hamood Abdul N.
Published: December 2019

We previously identified a novel regulator of the exotoxin A gene (
toxA
) in
Pseudomonas aeruginosa
, PtxR, that belongs to the LysR family of prokaryotic regulatory proteins. Preliminary data also suggest that PtxR affects the expression of siderophores in
P. aeruginosa
. Because
toxA
expression and siderophore production in this organism are coordinately regulated by the ferric uptake regulator (Fur) and the Fur-regulated alternative sigma factor PvdS, regulation of
ptxR
itself in the context of these regulators was examined. RNase protection analyses of
ptxR
transcription revealed that there are two independent transcription initiation sites (T1 and T2). While transcription from the promoter of T1 is constitutive throughout the growth cycle of PAO1, transcription from the second promoter (P2) is negatively affected by iron. Transcription from the P2 promoter is constitutive in a
fur
mutant under microaerobic conditions but still iron regulated during aerobic growth. High concentrations (>100 nM) of the ferric uptake regulatory protein (Fur) failed to bind to either of the promoter regions of
ptxR
in either gel mobility shift assays or DNase I footprint experiments. These results indicate that Fur indirectly regulates the iron-dependent expression of
ptxR
. Iron-regulated transcription of
ptxR
from the P2 promoter, but not constitutive expression from the P1 promoter, was dependent on the Fur-regulated alternative sigma factor gene
pvdS
, even under aerobic conditions. Consequently, there are two levels of iron-regulated expression of
ptxR
. The iron-regulated expression of
ptxR
under microaerobic conditions from the P2 promoter of
ptxR
is mediated indirectly by Fur through the iron-regulated expression of
pvdS
. In contrast,
pvdS
-mediated iron regulation of
ptxR
under aerobic conditions is Fur independent.
Purine Salvage in Two Halophilic Archaea: Characterization of Salvage Pathways and Isolation of Mutants Resistant to Purine Analogs
Authors: Stuer-Lauridsen Birgitte , Nygaard Per
Published: December 2019

In exponentially growing cultures of the extreme halophile
Halobacterium halobium
and the moderate halophile
Haloferax volcanii
, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed. This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-α-1-pyrophosphate) in archaea. The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays. The levels varied significantly between the two organisms. The most significant difference was the absence of GMP reductase activity in
H. halobium
. The metabolism of exogenous purines was investigated in growing cultures. Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids. Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis.
H. volcanii
was more sensitive than
H. halobium
, and purine base analogs were more toxic than nucleoside analogs. Growth of
H. volcanii
was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of
H. halobium
. Spontaneous mutants resistant to purine analogs were isolated. The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake. A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine. The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels. Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.
Mutational Analysis of UMP Kinase from
Escherichia coli
Authors: Bucurenci Nadia , Serina Lidia , Zaharia Cristina , Landais Stéphanie , Danchin Antoine , Bârzu Octavian
Published: December 2019

UMP kinase from
Escherichia coli
is one of the four regulatory enzymes involved in the de novo biosynthetic pathway of pyrimidine nucleotides. This homohexamer, with no counterpart in eukarya, might serve as a target for new antibacterial drugs. Although the bacterial enzyme does not show sequence similarity with any other known nucleoside monophosphate kinase, two segments between amino acids 35 to 78 and 145 to 194 exhibit 28% identity with phosphoglycerate kinase and 30% identity with aspartokinase, respectively. Based on these similarities, a number of residues of
E. coli
UMP kinase were selected for site-directed mutagenesis experiments. Biochemical, kinetic, and spectroscopic analysis of the modified proteins identified residues essential for catalysis (Asp146), binding of UMP (Asp174), and interaction with the allosteric effectors, GTP and UTP (Arg62 and Asp77).
Synthesis and Differential Turnover of the CYS3 Regulatory Protein of
Neurospora crassa
Are Subject to Sulfur Control
Authors: Tao Ying , Marzluf George A.
Published: December 2019

The transcription factor CYS3 of
Neurospora crassa
is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner.
cys-3
expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited.
cys-3
transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression,
cys-3
transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.
Characterization of Glucose-Specific Catabolite Repression-Resistant Mutants of
Bacillus subtilis
: Identification of a Novel Hexose:H
+
Symporter
Authors: Paulsen Ian T. , Chauvaux Sylvie , Choi Peter , Saier Milton H.
Published: December 2019

Insertional mutagenesis was conducted on
Bacillus subtilis
cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H
+
symporter of
Escherichia coli
and the glucose/galactose:H
+
symporter of
Brucella abortus
. In a wild-type
B. subtilis
genetic background, the
glcP
::Tn
10
mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [
14
C]glucose and [
14
C]methyl α-glucoside uptake. In a Δ
pts
genetic background no phenotype was observed, suggesting that expression of the
glcP
gene required a functional phosphotransferase system. When overproduced in a Δ
pts
mutant of
E. coli
, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that
B. subtilis
possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.