bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Organization of thednaARegion ofWolbachia
Authors: Sun Ling V. , Babaratsas Alekos , Savakis Charalambos , O’Neill Scott L. , Bourtzis Kostas
Published: December 2019

ThednaAregion ofWolbachia, an intracellular bacterial parasite of insects, is unique. AglnAcognate was found upstream of thednaAgene, while neither of the two open reading frames detected downstream ofdnaAhas any homologue in the database. This unusual gene arrangement may reflect requirements associated with the unique ecological niche this agent occupies.
The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto Are Secreted in Culture from Pseudomonas syringaePathovars via the Hrp (Type III) Protein Secretion System in a Temperature- and pH-Sensitive Manner
Authors: van Dijk Karin , Fouts Derrick E. , Rehm Amos H. , Hill Angela R. , Collmer Alan , Alfano James R.
Published: December 2019

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways fromPseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22°C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel theP. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name ofhrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.
Heterologous Expression and Characterization of the Purified Oxygenase Component of Rhodococcus globerulusP6 Biphenyl Dioxygenase and of Chimeras Derived from It
Authors: Chebrou Hervé , Hurtubise Yves , Barriault Diane , Sylvestre Michel
Published: December 2019

In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component ofRhodococcus globerulusP6 biphenyl dioxygenase. The α or β subunit of P6 oxygenase was exchanged with the corresponding subunit ofPseudomonassp. strain LB400 or ofComamonas testosteroniB-356 to create new chimeras that were purified ht-proteins and designated ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, ht-αLB400βP6, and ht-αB-356βP6. ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356were not expressed active in recombinantEscherichia colicells carrying P6bphA1andbphA2, P6bphA1and LB400bphE, or P6bphA1and B-356bphEbecause the [2Fe-2S] Rieske cluster of P6 oxygenase α subunit was not assembled correctly in these clones. On the other hand ht-αLB400βP6and ht-αB-356βP6were produced active inE. coli. Furthermore, active purified ht-αP6βP6, ht-αP6βLB400, ht-αP6βB-356, showing typical spectra for Rieske-type proteins, were obtained fromPseudomonas putidaKT2440 carrying constructions derived from the new shuttleE. coli-Pseudomonasvector pEP31, designed to produce ht-proteins inPseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an α and a β subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.
Catalytic Properties of the 3-Chlorocatechol-Oxidizing 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from Sphingomonas sp. Strain BN6
Authors: Riegert Ulrich , Heiss Gesche , Kuhm Andrea Elisabeth , Müller Claudia , Contzen Matthias , Knackmuss Hans-Joachim , Stolz Andreas
Published: December 2019

The 2,3-dihydroxybiphenyl dioxygenase fromSphingomonassp. strain BN6 (BphC1-BN6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. The turnover of different substrates and the effects of various inhibitors on BphC1-BN6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (BphC2-BN6) as well as with those of the archetypical catechol 2,3-dioxygenase (C23O-mt2) encoded by the TOL plasmid. Cell extracts containing C23O-mt2 or BphC2-BN6 converted the relevant substrates with an almost constant rate for at least 10 min, whereas BphC1-BN6 was inactivated significantly within the first minutes during the turnover of all substrates tested. Furthermore, BphC1-BN6 was much more sensitive than the other two enzymes to inactivation by the Fe(II) ion-chelating compoundo-phenanthroline. The reason for inactivation of BphC1-BN6 appeared to be the loss of the weakly bound ferrous ion, which is the cofactor in the catalytic center. A mutant enzyme of BphC1-BN6 constructed by site-directed mutagenesis showed a higher stability to inactivation byo-phenanthroline and an increased catalytic efficiency for the conversion of 2,3-dihydroxybiphenyl and 3-methylcatechol but was still inactivated during substrate oxidation.
The bvr Locus of Listeria monocytogenes Mediates Virulence Gene Repression by β-Glucosides
Authors: Brehm Klaus , Ripio María-Teresa , Kreft Jürgen , Vázquez-Boland José-Antonio
Published: December 2019

The β-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence geneshlyandplcAinListeria monocytogenesNCTC 7973. This led to the hypothesis that β-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes ifL. monocytogenesis living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressedhly. This suggested that the downregulation of virulence genes by β-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates ofL. monocytogenesby using the PrfA-dependent phospholipase C geneplcBas a reporter. Utilization of any fermentable sugar causedplcBrepression in wild-typeL. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973,plcBwas only repressed by β-glucosides. Thus, the regulation ofL. monocytogenesvirulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to β-glucosides. We have identified inL. monocytogenesa 4-kb operon,bvrABC, encoding an antiterminator of the BglG family (bvrA), a β-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from otherListeriaspp. Transcription ofbvrBwas induced by cellobiose and salicin but not by arbutin. Disruption of thebvroperon by replacing part ofbvrABwith an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that thebvrlocus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found inL. monocytogenesthat is involved in environmental regulation of virulence genes.
Essential Components of the Ti Plasmidtrb System, a Type IV Macromolecular Transporter
Authors: Li Pei-Li , Hwang Ingyu , Miyagi Heather , True Heather , Farrand Stephen K.
Published: December 2019

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production ofAgrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58ΔaccR mutations intrbB, -C, -D, -E, -L, -F, -G, and -Habolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbImutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation intraI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of thetrb genes except traI and trbJ can be complemented by functions coded for by pAtC58.
Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria
Authors: Hu Ping , Leighton Terrance , Ishkhanova Galina , Kustu Sydney
Published: December 2019

Previous studies showed thatSalmonella typhimuriumapparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation inBacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size ofB. subtilisclearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious inB. subtilis. To rule out the possibility that differences were due to the fact thatB. subtilishas only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase inSalmonella. Studies of theS. typhimurium gdhstrain in ammonia-limited continuous culture and ofgdh glnAdouble-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results withB. subtilisare that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.
Functional and Transcriptional Analyses of a Fengycin Synthetase Gene, fenC, from Bacillus subtilis
Authors: Lin Tsuey-Pin , Chen Chyi-Liang , Chang Li-Kwan , Tschen Johannes Scheng-Ming , Liu Shih-Tung
Published: December 2019

A 37-kb DNA fragment containing five fengycin synthetase genes, includingfenC,fenD,fenE,fenA, andfenB, was cloned and sequenced. Among these genes,fenCencodes a fengycin synthetase 2,560 amino acids long with an estimated molecular mass of 287 kDa. This protein contains two amino acid activation modules, FenC1 and FenC2, which activatel-glutamic acid andl-ornithine, respectively. Primer extension, using mRNA isolated from the log-phase cells, identified a transcription start site located 86 nucleotides upstream from the initiation codon offenC, implying that a promoter is located upstream from the start site. Primer extension using total RNA isolated from stationary-phase cells also identified a transcription start site located 61 nucleotides upstream from the initiation codon offenC. Gene fusion studies demonstrated that in nHA medium, the cells transcribe the fengycin synthetase genes at two different stages of cell growth. The promoter is active during the log phase, and the activity reaches the highest level during the late log phase. The activity decreases sharply but is maintained at a low level for approximately 24 h after cells enter the early stationary phase. The results of this investigation also suggest that the transcription offenCis positively regulated during the late log phase. Results presented herein provide further insight into fengycin synthesis byB. subtilisF29-3.
In Vitro Analysis of the Butyrolactone Autoregulator Receptor Protein (FarA) of Streptomyces lavendulae FRI-5 Reveals that FarA Acts as a DNA-Binding Transcriptional Regulator That Controls Its Own Synthesis
Authors: Kitani Shigeru , Kinoshita Hiroshi , Nihira Takuya , Yamada Yasuhiro
Published: December 2019

FarA of Streptomyces lavendulae FRI-5 is a specific receptor protein for IM-2, a butyrolactone autoregulator that controls the production of a blue pigment and the nucleoside antibiotics showdomycin and minimycin. Gel shift assays demonstrated that FarA binds to the farA upstream region and that this binding is abolished in the presence of IM-2. The FarA binding sequence was localized by DNase I footprinting to a 28-bp sequence located approximately 70 bp upstream of the farA translational start site. High-resolution S1 nuclease mapping of farAtranscripts revealed a putative transcription start site, located at an A residue positioned 64 bp upstream from the farAtranslation start codon and 4 bp downstream from an Escherichia coli ς70-like −10 recognition region. The FarA-binding sequence overlaps this −10 region and contains thefarA transcription initiation site, suggesting that FarA acts as a repressor that, in the absence of IM-2, represses transcription of farA.
Cloning and Analysis of the rnc-era-recOOperon from Pseudomonas aeruginosa
Authors: Powell Bradford , Peters Howard K. , Nakamura Yoshikazu , Court Donald
Published: December 2019

The rnc operon from Pseudomonas aeruginosahas been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of thernc, era, and recO genes inEscherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of eramutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog.
Reduction of Adenosine-5′-Phosphosulfate Instead of 3′-Phosphoadenosine-5′-Phosphosulfate in Cysteine Biosynthesis by
Rhizobium meliloti
and Other Members of the Family
Rhizobiaceae
Authors: Abola A. Pia , Willits Michael G. , Wang Richard C. , Long Sharon R.
Published: December 2019

We have cloned and sequenced three genes from
Rhizobium meliloti
(
Sinorhizobium meliloti
) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the
Escherichia coli cysDN
genes, which code for an ATP sulfurylase (EC
2.7.7.4
). The third gene has homology to the
E. coli cysH
gene, a 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase (EC
1.8.99.4
), but has greater homology to a set of genes found in
Arabidopsis thaliana
that encode an adenosine-5′-phosphosulfate (APS) reductase. In order to determine the specificity of the
R. meliloti
reductase, the
R. meliloti cysH
homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the
A. thaliana
reductases, the histidine-tagged
R. meliloti cysH
gene product appears to favor APS over PAPS as a substrate, with a

K
m

for APS of 3 to 4 μM but a

K
m

for PAPS of >100 μM. In order to determine whether this preference for APS is unique to
R. meliloti
among members of the family
Rhizobiaceae
or is more widespread, cell extracts from
R. leguminosarum
,
Rhizobium
sp. strain NGR234,
Rhizobium fredii
(
Sinorhizobium fredii
), and
Agrobacterium tumefaciens
were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.
The
napF
and
narG
Nitrate Reductase Operons in
Escherichia coli
Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite
Authors: Wang Henian , Tseng Ching-Ping , Gunsalus Robert P.
Published: December 2019

Escherichia coli
synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the
narGHJI
operon and a periplasmic cytochrome
c
-linked nitrate reductase encoded by the
napFDAGHBC
operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine
napF
and
narG
gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the
napF-lacZ
and
narG-lacZ
reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The
napF
operon apparently encodes a “low-substrate-induced” reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the
narGHJI
operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The
narGHJI
operon is therefore a “high-substrate-induced” operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of
napF
gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of
narG
and
napF
expression must be reconsidered.
A New Class of Cobalamin Transport Mutants (
btuF
) Provides Genetic Evidence for a Periplasmic Binding Protein in
Salmonella typhimurium
Authors: Van Bibber Michael , Bradbeer Clive , Clark Nica , Roth John R.
Published: December 2019

No periplasmic binding protein has been demonstrated for the ATP-binding cassette (ABC)-type cobalamin transporter BtuCD. New mutations (
btuF
) are described that affect inner-membrane transport. The BtuF protein has a signal sequence and resembles the periplasmic binding proteins of several other ABC transporters.
Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in
Escherichia coli
K-12
Authors: Atlung T. , Hansen F. G.
Published: December 2019

Expression of the
dnaA
gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14°C than at 37°C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30°C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of
dnaA
gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.
Involvement of the
cis/trans
Isomerase Cti in Solvent Resistance of
Pseudomonas putida
DOT-T1E
Authors: Junker Frank , Ramos Juan L.
Published: December 2019

Pseudomonas putida
DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the
cti
gene of this strain, which encodes the
cis/trans
isomerase, termed Cti, that catalyzes the
cis-trans
isomerization of esterified fatty acids in phospholipids, mainly
cis
-oleic acid (C
16:1,9
) and
cis
-vaccenic acid (C
18:1,11
), in response to solvents. To determine the importance of this
cis/trans
isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5°C, the Cti-null mutant strain grew significantly slower at temperatures above 37°C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the
cti
gene is present in all the tested
P. putida
strains, including non-solvent-resistant ones, and in some other
Pseudomonas
species.
Analysis of Fusion Junctions of Circularized Chromosomes in
Streptomyces griseus
Authors: Kameoka Daisuke , Lezhava Alexander , Zenitani Hiroyuki , Hiratsu Keiichiro , Kawamoto Makoto , Goshi Kohei , Inada Kuninobu , Shinkawa Hidenori , Kinashi Haruyasu
Published: December 2019

A filamentous soil bacterium,
Streptomyces griseus
2247, carries a 7.8-Mb linear chromosome. We previously showed by macrorestriction analysis that mutagenic treatments easily caused deletions at both ends of its linear chromosome and changed the chromosome to a circular form. In this study, we confirmed chromosomal circularization by cloning and sequencing the junction fragments from two deletion mutants, 404-23 and N2. The junction sequences were compared with the corresponding right and left deletion end sequences in the parent strain, 2247. No homology and a 6-bp microhomology were found between the two deletion ends of the 404-23 and N2 mutants, respectively, which indicate that the chromosomal circularization was caused by illegitimate recombination without concomitant amplification. The circularized chromosomes were stably maintained in both mutants. Therefore, the chromosomal circularization might have occurred to prevent lethal deletions, which otherwise would progress into the indispensable central regions of the chromosome.
The
Caulobacter crescentus
CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties
Authors: Lin Bin , Covalle Kelly L. , Maddock Janine R.
Published: December 2019

The
Caulobacter crescentus
CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30°C and displayed equilibrium binding constants of 1.2 and 0.5 μM, respectively, in the presence of Mg
2+
. In the absence of Mg
2+
, the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively.
N
-Methyl-3′-
O
-anthranoyl (mant)–guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg
2+
was extremely rapid (

k
d

= 1.4 and 1.5 s
−1
, respectively), 10
3
- to 10
5
-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg
2+
. Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 × 10
−4
s
−1
corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.
A Sequence Downstream of the Initiation Codon Is Essential for Cold Shock Induction of
cspB
of
Escherichia coli
Authors: Etchegaray Jean-Pierre , Inouye Masayori
Published: December 2019

Cold shock induction of
cspB
has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of
cspB
at low temperature also requires the translational
cis
-acting element called the downstream box (DB). Full induction of
cspB
at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.
Role of CCAA Nucleotide Repeats in Regulation of Hemoglobin and Hemoglobin-Haptoglobin Binding Protein Genes of
Haemophilus influenzae
Authors: Ren Zhen , Jin Hongfan , Whitby Paul W. , Morton Daniel J. , Stull Terrence L.
Published: December 2019

Haemophilus influenzae
utilizes hemoglobin and hemoglobin-haptoglobin as heme sources. The
H. influenzae
hemoglobin- and hemoglobin-haptoglobin binding protein genes,
hgpA
,
hgpB
, and
hgpC
, contain lengths of tetrameric CCAA repeats. Using an
hgpA-lacZ
translational gene fusion, we demonstrate phase-variable expression of
lacZ
associated with alteration in the length of the CCAA repeat region.
Expression of the
Staphylococcus aureus
UDP-
N
-Acetylmuramoyl-
l
-Alanyl-
d
-Glutamate:
l
-Lysine Ligase in
Escherichia coli
and Effects on Peptidoglycan Biosynthesis and Cell Growth
Authors: Mengin-Lecreulx Dominique , Falla Tim , Blanot Didier , van Heijenoort Jean , Adams David J. , Chopra Ian
Published: December 2019

The monomer units in the
Escherichia coli
and
Staphylococcus aureus
cell wall peptidoglycans differ in the nature of the third amino acid in the
l
-alanyl-γ-
d
-glutamyl-X-
d
-alanyl-
d
-alanine side chain, where X is
meso
-diaminopimelic acid or
l
-lysine, respectively. The
murE
gene from
S. aureus
encoding the UDP-
N
-acetylmuramoyl-
l
-alanyl-
d
-glutamate:
l
-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in
E. coli
rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the
meso
-diaminopimelic acid residues were replaced by
l
-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively
meso
-diaminopimelic acid, suggesting that no transpeptidation could occur between the ɛ-amino group of
l
-lysine and the α-carboxyl group of
d
-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of
meso
-diaminopimelic acid- and
l
-lysine-containing peptidoglycan precursors confirmed the presence of
l
-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.
Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential
uppS
Gene
Authors: Apfel Christian M. , Takács Béla , Fountoulakis Michael , Stieger Martin , Keck Wolfgang
Published: December 2019

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-
trans
,poly-
cis
-decaprenylcistransferase; EC
2.5.1.31
) was purified from the soluble fraction of
Escherichia coli
by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (
E
,
E
)-[1-
3
H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphos-phate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all
E. coli
proteins yielded a single match with a protein of unassigned function (SWISS-PROT
Q47675
; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in
Saccharomyces cerevisiae
, and in
Caenorhabditis elegans
. The homologous genes (
uppS
) were cloned from
E. coli
,
Haemophilus influenzae
, and
Streptococcus pneumoniae
, expressed in
E. coli
as amino-terminal His-tagged fusion proteins, and purified over a Ni
2+
affinity column. An untagged version of the
E. coli uppS
gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged
E. coli
Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl
2
dependent. With the use of a regulatable gene disruption system, we demonstrated that
uppS
is essential for growth in
S. pneumoniae
R6.
A Second Operator Is Involved in
Pseudomonas aeruginosa
Elastase (
lasB
) Activation
Authors: Anderson Ronda M. , Zimprich Chad A. , Rust Lynn
Published: December 2019

Pseudomonas aeruginosa
LasB elastase gene (
lasB
) transcription is controlled by the two-component quorum-sensing system of LasR, and the autoinducer, 3OC
12
-HSL (
N
-3-[oxododecanoyl]homoserine lactone). LasR and 3OC
12
-HSL-mediated
lasB
activation requires a functional operator sequence (OP1) in the
lasB
promoter region. Optimal activation of
lasB
, however, requires a second sequence of 70% identity to OP1, named OP2, located 43 bp upstream of OP1. In this study, we used sequence substitutions and insertion mutations in
lasB
p-
lacZ
fusion plasmids to explore the role of OP2 in
lasB
activation. Our results demonstrate that (i) OP1 and OP2 synergistically mediate
lasB
activation; (ii) OP2, like OP1, responds to LasR and 3OC
12
-HSL; and (iii) the putative autoinducer-binding domain of LasR is not required for synergistic activation from OP1 and OP2.
Induction of β-Lactamase Influences the Course of Development in
Myxococcus xanthus
Authors: O’Connor Kathleen A. , Zusman David R.
Published: December 2019

Myxococcus xanthus
is a gram-negative bacterium that develops in response to starvation on a solid surface. The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores. Previously, we have shown that the induction of β-lactamase is associated with starvation-independent sporulation in liquid culture (K. A. O’Connor and D. R. Zusman, Mol. Microbiol. 24:839–850, 1997). In this paper, we show that the chromosomally encoded β-lactamase of
M. xanthus
is autogenously induced during development. The specific activity of the enzyme begins to increase during aggregation, before spores are detectable. The addition of inducers of β-lactamase in
M. xanthus
, such as ampicillin,
d
-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells. In addition, the exogenous induction of β-lactamase allows
M. xanthus
to fruit on media containing concentrations of nutrients that are normally too high to support development. We propose that the induction of β-lactamase is an integral step in the development of
M. xanthus
and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores. In support of this hypothesis, we show that exogenous induction of β-lactamase can rescue aggregation and sporulation of certain mutants. Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy. These results show that the signal transduction pathway leading to the induction of β-lactamase plays an important role in aggregation and sporulation in
M. xanthus
.
Ferric Enterochelin Transport in
Yersinia enterocolitica
: Molecular and Evolutionary Aspects
Authors: Schubert S. , Fischer D. , Heesemann J.
Published: December 2019

Yersinia enterocolitica
is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the
Yersinia fep-fes
gene cluster orthologous to the
Escherichia coli
ferrienterochelin transport genes (
fepA
,
fepDGC
, and
fepB
) and the esterase gene
fes
. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by
fepC
and
fes
, respectively. A frameshift mutation within the
fepA
gene led to expression of a truncated polypeptide of 40 kDa. The
fepD
,
fepG
, and
fes
genes of
Y. enterocolitica
were shown to complement corresponding
E. coli
mutants. Insertional mutagenesis of
fepD
or
fes
genes abrogates enterochelin-supported growth of
Y. enterocolitica
on iron-chelated media. In contrast to
E. coli
, the
fep-fes
gene cluster in
Y. enterocolitica
consists solely of genes required for uptake and utilization of enterochelin (
fep
) and not of enterochelin synthesis genes such as
entF
. By Southern hybridization,
fepDGC
and
fes
sequences could be detected in
Y. enterocolitica
biotypes IB, IA, and II but not in biotype IV strains,
Yersinia pestis
, and
Yersinia pseudotuberculosis
strains. According to sequence alignment data and the coherent structure of the
Yersinia fep-fes
gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family
Enterobacteriaceae
.
Initial Reactions in Anaerobic Oxidation of
m
-Xylene by the Denitrifying Bacterium
Azoarcus
sp. Strain T
Authors: Krieger Cynthia J. , Beller Harry R. , Reinhard Martin , Spormann Alfred M.
Published: December 2019

The initial enzymatic steps in anaerobic
m
-xylene oxidation were studied in
Azoarcus
sp. strain T, a denitrifying bacterium capable of mineralizing
m
-xylene via 3-methylbenzoate. Permeabilized cells of
m
-xylene-grown
Azoarcus
sp. strain T catalyzed the addition of
m
-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to
E
-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of
m
-xylene-grown
Azoarcus
sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo
m
-xylene consumption. Based on these findings, we propose that
Azoarcus
sp. strain T anaerobically oxidizes
m
-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and
E
-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.
Efficiency of Transcription from Promoter Sequence Variants in
Lactobacillus
Is Both Strain and Context Dependent
Authors: McCracken Andrea , Timms Peter
Published: December 2019

The introduction of consensus −35 (TTGACA) and −10 (TATAAT) hexamers and a TG motif into the
Lactobacillus acidophilus
ATCC 4356 wild-type
slpA
promoter resulted in significant improvements (4.3-, 4.1-, and 10.7-fold, respectively) in transcriptional activity in
Lactobacillus fermentum
BR11. In contrast, the same changes resulted in decreased transcription in
Lactobacillus rhamnosus
GG. The TG motif was shown to be important in the context of weak −35 and −10 hexamers (
L. fermentum
BR11) or a consensus −10 hexamer (
L. rhamnosus
GG). Thus, both strain- and context-dependent effects are critical factors influencing transcription in
Lactobacillus.
Molecular Evolution of the Pathogenicity Island of Enterotoxigenic
Bacteroides fragilis
Strains
Authors: Franco Augusto A. , Cheng Rodney K. , Chung Gyung-Tae , Wu Shaoguang , Oh Hee-Bok , Sears Cynthia L.
Published: December 2019

Enterotoxigenic
Bacteroides fragilis
(ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic
B. fragilis
(NTBF) strains, we found that
bft
and a second metalloprotease gene (
mpII
) are contained in an ∼6-kb pathogenicity island (termed
B. fragilis
pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (
bfmA
and
bfmB
) organized in one operon and a putative origin of transfer (
oriT
) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (
bfmC
) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from
Escherichia coli
. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that
bfmB
and
bfmC
are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the
bfmB
and
bfmC
stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the
B. fragilis
chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.
Bacterial Phylogenetic Clusters Revealed by Genome Structure
Authors: Liu Shu-Lin , Schryvers Anthony B. , Sanderson Kenneth E. , Johnston Randal N.
Published: December 2019

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-
Ceu
I, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera:
Salmonella
and
Pasteurella
. Among
Salmonella
spp., I-
Ceu
I mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the
Salmonella
spp. In marked contrast, the
Pasteurella
strains have very different genome structures among and even within individual species. The divergence of
Pasteurella
was also reflected in 16S rDNA sequences and far exceeded that seen between
Escherichia
and
Salmonella
. Based on this diversity, the
Pasteurella haemolytica
strains we analyzed could be divided into 14 phylogenetic groups and the
Pasteurella multocida
strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for
Salmonella
and
Escherichia coli
were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of
P. multocida
and
P. haemolytica
to be divided into different species, genera, or even higher ranks. On the other hand, strains of
Pasteurella ureae
and
Pasteurella pneumotropica
are very similar to those of
P. multocida
in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-
Ceu
I-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.
Anaerobic Growth of
Paracoccus denitrificans
Requires Cobalamin: Characterization of
cobK
and
cobJ
Genes
Authors: Shearer Neil , Hinsley Andrew P. , Van Spanning Rob J. M. , Spiro Stephen
Published: December 2019

A pleiotropic mutant of
Paracoccus denitrificans
, which has a severe defect that affects its anaerobic growth when either nitrate, nitrite, or nitrous oxide is used as the terminal electron acceptor and which is also unable to use ethanolamine as a carbon and energy source for aerobic growth, was isolated. This phenotype of the mutant is expressed only during growth on minimal media and can be reversed by addition of cobalamin (vitamin B
12
) or cobinamide to the media or by growth on rich media. Sequence analysis revealed the mutation causing this phenotype to be in a gene homologous to
cobK
of
Pseudomonas denitrificans
, which encodes precorrin-6x reductase of the cobalamin biosynthesis pathway. Convergently transcribed with
cobK
is a gene homologous to
cobJ
of
Pseudomonas denitrificans
, which encodes precorrin-3b methyltransferase. The inability of the cobalamin auxotroph to grow aerobically on ethanolamine implies that wild-type
P. denitrificans
(which can grow on ethanolamine) expresses a cobalamin-dependent ethanolamine ammonia lyase and that this organism synthesizes cobalamin under both aerobic and anaerobic growth conditions. Comparison of the
cobK
and
cobJ
genes with their orthologues suggests that
P. denitrificans
uses the aerobic pathway for cobalamin synthesis. It is paradoxical that under anaerobic growth conditions,
P. denitrificans
appears to use the aerobic (oxygen-requiring) pathway for cobalamin synthesis. Anaerobic growth of the cobalamin auxotroph could be restored by the addition of deoxyribonucleosides to minimal media. These observations provide evidence that
P. denitrificans
expresses a cobalamin-dependent ribonucleotide reductase, which is essential for growth only under anaerobic conditions.
Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis in
Streptomyces coelicolor
A3(2) as a DNA-Binding Protein
Authors: Arias Paloma , Fernández-Moreno Miguel A. , Malpartida Francisco
Published: December 2019

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the
act
cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type
act
II-ORF4 gene but not with the
act
II-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from
Escherichia coli
cultures; both showed specific DNA-binding activity for the
act
VI-ORF1–ORFA and
act
III-
act
I intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the −35 regions of the corresponding promoters, showing the consensus sequence 5′-TCGAG-3′. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the
act
genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the
act
biosynthetic genes.
Characterization of Activity and Expression of Isocitrate Lyase in
Mycobacterium avium
and
Mycobacterium tuberculosis
Authors: Höner Zu Bentrup Kerstin , Miczak Andras , Swenson Dana L. , Russell David G.
Published: December 2019

Analysis by two-dimensional gel electrophoresis revealed that
Mycobacterium avium
expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the
M. tuberculosis
database (Sanger) with similarity to the enzyme isocitrate lyase of both
Corynebacterium glutamicum
and
Rhodococcus fascians
. Only marginal similarity was observed between this open reading frame (ORF) (termed
icl
) and a second distinct ORF (named
aceA
) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both
M. avium
and
M. tuberculosis
the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.