bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Characterization of a Tetracycline Resistance Determinant Present in
Agrobacterium tumefaciens
C58
Authors: Luo Zhao-Qing , Farrand Stephen K.
Published: December 2019

Agrobacterium tumefaciens
C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in
Escherichia coli
and
Agrobacterium
was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes,
tetA
and
tetR
, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant,
tetR
was disrupted by an IS
426
. The homologous region from wild-type NT1 contained an intact
tetR
gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus
Agrobacterium
surveyed, only strains C58 and T37 contained the
tet
determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this
tet
gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this
tet
gene system. The divergent promoter region of this
tet
system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the
tet
determinant from the IncP1α R plasmid RP4. TetR repressor proteins from the
Agrobacterium tet
system and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetR
C58
) on the
tetA
gene from strain RP4 (
tetA
RP4
) was not relieved by tetracycline, repression of
tetA
C58
by TetR
RP4
was lifted by this antibiotic.
A Plant-Type (β-Class) Carbonic Anhydrase in the Thermophilic MethanoarchaeonMethanobacterium thermoautotrophicum
Authors: Smith Kerry S. , Ferry James G.
Published: December 2019

Carbonic anhydrase, a zinc enzyme catalyzing the interconversion of carbon dioxide and bicarbonate, is nearly ubiquitous in the tissues of highly evolved eukaryotes. Here we report on the first known plant-type (β-class) carbonic anhydrase in the archaea. TheMethanobacterium thermoautotrophicumΔHcabgene was hyperexpressed inEscherichia coli, and the heterologously produced protein was purified 13-fold to apparent homogeneity. The enzyme, designated Cab, is thermostable at temperatures up to 75°C. No esterase activity was detected withp-phenylacetate as the substrate. The enzyme is an apparent tetramer containing approximately one zinc per subunit, as determined by plasma emission spectroscopy. Cab has a CO2hydration activity with akcatof 1.7 × 104s−1andKmfor CO2of 2.9 mM at pH 8.5 and 25°C. Western blot analysis indicates that Cab (β class) is expressed inM. thermoautotrophicum; moreover, a protein cross-reacting to antiserum raised against the γ carbonic anhydrase fromMethanosarcina thermophilawas detected. These results show that β-class carbonic anhydrases extend not only into theArchaeadomain but also into the thermophilic prokaryotes.
Characterization of MexT, the Regulator of the MexE-MexF-OprN Multidrug Efflux System of
Pseudomonas aeruginosa
Authors: Köhler Thilo , Epp Simone F. , Curty Lasta Kocjancic , Pechère Jean-Claude
Published: December 2019

We investigated the regulation of the MexEF-OprN multidrug efflux system of
Pseudomonas aeruginosa
, which is overexpressed in
nfxC
-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the
mexEF-oprN
operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in
Escherichia coli
from a plasmid carrying the
mexT
gene. Expression of a
mexE
::
lacZ
fusion was 10-fold higher in
nfxC
-type mutants than in the wild-type strain; however, transcription of
mexT
as well as the
mexT
DNA region was unchanged. Located adjacent to
mexT
but transcribed in opposite direction, the beginning of an ORF termed
qrh
(quinone oxidoreductase homologue) was identified. Expression of a
qrh
::
lacZ
fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of
nfxC
-type mutants to carbapenem antibiotics.
In Vitro Characterization of Peptidoglycan-Associated Lipoprotein (PAL)–Peptidoglycan and PAL–TolB Interactions
Authors: Bouveret Emmanuelle , Bénédetti Hélène , Rigal Alain , Loret Erwann , Lazdunski Claude
Published: December 2019

The Tol-peptidoglycan-associated lipoprotein (PAL) system of
Escherichia coli
is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.
Hybrid Motor with H
+
- and Na
+
-Driven Components Can Rotate
Vibrio
Polar Flagella by Using Sodium Ions
Authors: Asai Yukako , Kawagishi Ikuro , Sockett R. Elizabeth , Homma Michio
Published: December 2019

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium
Vibrio alginolyticus
is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium
Rhodobacter sphaeroides
. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA.
R. sphaeroides
MotB has high similarity only to the transmembrane region of PomB. To examine whether the
R. sphaeroides
motor genes can function in place of the
pomA
and
pomB
genes of
V. alginolyticus
, we constructed plasmids including both
motA
and
motB
or
motA
alone and transformed them into missense and null
pomA
-paralyzed mutants of
V. alginolyticus
. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand,
pomB
mutants were not restored to motility by any plasmid containing
motA
and/or
motB
. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of
R. sphaeroides
can generate torque by coupling with the sodium ion flux in place of PomA of
V. alginolyticus.
Pathogenic
Yersinia
Species Carry a Novel, Cold-Inducible Major Cold Shock Protein Tandem Gene Duplication Producing both Bicistronic and Monocistronic mRNA
Authors: Neuhaus Klaus , Francis Kevin P. , Rapposch Sonja , Görg Angelika , Scherer Siegfried
Published: December 2019

Inverse PCR was used to amplify major cold shock protein (MCSP) gene families from a diverse range of bacteria, including the psychrotolerant
Yersinia enterocolitica
, which was found to have two almost identical MCSP coding regions (
cspA1
and
cspA2
) located approximately 300 bp apart. This tandem gene duplication was also found in
Y. pestis
,
Y. pseudotuberculosis
, and
Y. ruckeri
but not in other bacteria. Analysis of the transcriptional regulation of this MCSP gene in
Y. enterocolitica
, performed by using both reverse transcriptase-PCR and Northern blot assays, showed there to be two cold-inducible mRNA templates arising from this locus: a monocistronic template of approximately 450 bp (
cspA1
) and a bicistronic template of approximately 900 bp (
cspA1/A2
). The former may be due to a secondary structure between
cspA1
and
cspA2
causing either 3′ degradation protection of
cspA1
or, more probably, partial termination after
cspA1
. Primer extension experiments identified a putative transcriptional start site (+1) which is flanked by a cold-box motif and promoter elements (−10 and −35) similar to those found in
Escherichia coli
cold-inducible MCSP genes. At 30°C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10°C, transcription of the bicistronic mRNA was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was apparent after only 10 min of cold shock from 30°C to 10°C. The data demonstrate an extraordinary capacity of the psychrotolerant
Y. enterocolitica
to produce major cold shock proteins upon cold shock.
A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in
Aspergillus fumigatus
Authors: Tsai Huei-Fung , Wheeler Michael H. , Chang Yun C. , Kwon-Chung K. J.
Published: December 2019

Aspergillus fumigatus
, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of
A. fumigatus
significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in
A. fumigatus
. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of
alb1
(for “albino 1”),
arp1
(for “aspergillus reddish-pink 1”), and
arp2
have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The
abr1
gene (for “aspergillus brown 1”) encodes a putative protein possessing two signatures of multicopper oxidases. The
abr2
gene product has homology to the laccase encoded by the
yA
gene of
Aspergillus nidulans
. The function of
ayg1
(for “aspergillus yellowish-green 1”) remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in
A. fumigatus
. The presence of a DHN-melanin pathway in
A. fumigatus
was supported by the accumulation of scytalone and flaviolin in the
arp1
deletant, whereas only flaviolin was accumulated in the
arp2
deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in
A. fumigatus
. However, the presence of
abr1
,
abr2
, and
ayg1
in addition to
alb1
,
arp1
, and
arp2
suggests that conidial pigment biosynthesis in
A. fumigatus
is more complex than the known DHN-melanin pathway.
Sulfide-Quinone Reductase from
Rhodobacter capsulatus
: Requirement for Growth, Periplasmic Localization, and Extension of Gene Sequence Analysis
Authors: Schütz Michael , Maldener Iris , Griesbeck Christoph , Hauska Günter
Published: December 2019

The entire sequence of the 3.5-kb fragment of genomic DNA from
Rhodobacter capsulatus
which contains the
sqr
gene and a second complete and two further partial open reading frames has been determined. A correction of the previously published
sqr
gene sequence (M. Schütz, Y. Shahak, E. Padan, and G. Hauska, J. Biol. Chem. 272:9890–9894, 1997) which in the deduced primary structure of the sulfide-quinone reductase changes four positive into four negative charges and the number of amino acids from 425 to 427 was necessary. The correction has no further bearing on the former sequence analysis. Deletion and interruption strains document that sulfide-quinone reductase is essential for photoautotrophic growth on sulfide. The sulfide-oxidizing enzyme is involved in energy conversion, not in detoxification. Studies with an alkaline phosphatase fusion protein reveal a periplasmic localization of the enzyme. Exonuclease treatment of the fusion construct demonstrated that the C-terminal 38 amino acids of sulfide-quinone reductase were required for translocation. An N-terminal signal peptide for translocation was not found in the primary structure of the enzyme. The possibility that the neighboring open reading frame, which contains a double arginine motif, may be involved in translocation has been excluded by gene deletion (rather, the product of this gene functions in an ATP-binding cassette transporter system, together with the product of one of the other open reading frames). The results lead to the conclusion that the sulfide-quinone reductase of
R. capsulatus
functions at the periplasmic surface of the cytoplasmic membrane and that this flavoprotein is translocated by a hitherto-unknown mechanism.
Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases inArchaea
Authors: Cann Isaac K. O. , Ishino Sonoko , Hayashi Ikuko , Komori Kayoko , Toh Hiroyuki , Morikawa Kosuke , Ishino Yoshizumi
Published: December 2019

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domainEucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote,Pyrococcus furiosus, expressed it inEscherichia coli, and characterized the biochemical properties of the gene product. The proteinPfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed thatPfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (α-like) DNA polymerases, while Pol II is a heterodimer.PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases inP. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domainsBacteria,Eucarya, andArchaea. The stimulatory effect ofPfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.
Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of
Escherichia coli
Central Carbon Metabolism
Authors: Sauer Uwe , Lasko Daniel R. , Fiaux Jocelyne , Hochuli Michel , Glaser Ralf , Szyperski Thomas , Wüthrich Kurt , Bailey James E.
Published: December 2019

The response of
Escherichia coli
central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional
13
C-
1
H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of
E. coli
strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in
E. coli
cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.
Cloning and Sequencing of a Novel
meta
-Cleavage Dioxygenase Gene Whose Product Is Involved in Degradation of γ-Hexachlorocyclohexane in
Sphingomonas paucimobilis
Authors: Miyauchi Keisuke , Adachi Yugo , Nagata Yuji , Takagi Masamichi
Published: December 2019

Sphingomonas
(formerly
Pseudomonas
)
paucimobilis
UT26 utilizes γ-hexachlorocyclohexane (γ-HCH), a halogenated organic insecticide, as a sole source of carbon and energy. In a previous study, we showed that γ-HCH is degraded to chlorohydroquinone (CHQ) and then to hydroquinone (HQ), although the rate of reaction from CHQ to HQ was slow (K. Miyauchi, S. K. Suh, Y. Nagata, and M. Takagi, J. Bacteriol. 180:1354–1359, 1998). In this study, we cloned and characterized a gene, designated
linE
, which is located upstream of
linD
and is directly involved in the degradation of CHQ. The LinE protein consists of 321 amino acids, and all of the amino acids which are reported to be essential for the activity of
meta
-cleavage dioxygenases are conserved in LinE.
Escherichia coli
overproducing LinE could convert both CHQ and HQ, producing γ-hydroxymuconic semialdehyde and maleylacetate, respectively, with consumption of O
2
but could not convert catechol, which is one of the major substrates for
meta
-cleavage dioxygenases. LinE seems to be resistant to the acylchloride, which is the ring cleavage product of CHQ and which seems to react with water to be converted to maleylacetate. These results indicated that LinE is a novel type of
meta
-cleavage dioxygenase, designated (chloro)hydroquinone 1,2-dioxygenase, which cleaves aromatic rings with two hydroxyl groups at
para
positions preferably. This study represents a direct demonstration of a new type of ring cleavage pathway for aromatic compounds, the hydroquinone pathway.
Role of Crotonyl Coenzyme A Reductase in Determining the Ratio of Polyketides Monensin A and Monensin B Produced by
Streptomyces cinnamonensis
Authors: Liu Haibin , Reynolds Kevin A.
Published: December 2019

The
ccr
gene, encoding crotonyl coenzyme A (CoA) reductase (CCR), was cloned from
Streptomyces cinnamonensis
C730.1 and shown to encode a protein with 90% amino acid sequence identity to the CCRs of
Streptomyces collinus
and
Streptomyces coelicolor
. A
ccr
-disrupted mutant,
S. cinnamonensis
L1, was constructed by inserting the
hyg
resistance gene into a unique
Bgl
II site within the
ccr
coding region. By use of the
ermE
* promoter, the
S. collinus ccr
gene was expressed from plasmids in
S. cinnamonensis
C730.1/pHL18 and L1/pHL18. CCR activity in mutant L1 was shown to decrease by more than 90% in both yeast extract-malt extract (YEME) medium and a complex fermentation medium, compared to that in wild-type C730.1. Compared to C730.1, mutants C730.1/pHL18 and L1/pHL18 exhibited a huge increase in CCR activity (14- and 13-fold, respectively) in YEME medium and a moderate increase (3.7- and 2.7-fold, respectively) in the complex fermentation medium. In the complex fermentation medium,
S. cinnamonensis
L1 produced monensins A and B in a ratio of 12:88, dramatically lower than the 50:50 ratio observed for both C730.1 and C730.1/pHL18. Plasmid (pHL18)-based expression of the
S. collinus ccr
gene in mutant L1 increased the monensin A/monensin B ratio to 42:58. Labeling experiments with [1,2-
13
C
2
]acetate demonstrated the same levels of intact incorporation of this material into the butyrate-derived portion of monensin A in both C730.1 and mutant C730.1/pLH18 but a markedly decreased level of such incorporation in mutant L1. The addition of crotonic acid at 15 mM led to significant increases in the monensin A/monensin B ratio in C730.1 and C730.1/pHL18 but had no effect in
S. cinnamonensis
L1. These results demonstrate that CCR plays a significant role in providing butyryl-CoA for monensin A biosynthesis and is present in wild-type
S. cinnamonensis
C730.1 at a level sufficient that the availability of the appropriate substrate (crotonyl-CoA) is limiting.
Random Transposition by Tn
916
in
Desulfitobacterium dehalogenans
Allows for Isolation and Characterization of Halorespiration-Deficient Mutants
Authors: Smidt Hauke , Song Donglin , van der Oost John , de Vos Willem M.
Published: December 2019

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium
Desulfitobacterium dehalogenans
, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn
916
. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of
D. dehalogenans
was isolated and mated with
Enterococcus faecalis
JH2-2 carrying Tn
916
. Insertion of one or two copies of Tn
916
into the chromosome of
D. dehalogenans
was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile
D. dehalogenans
have been disrupted.
d
-Allose Catabolism of
Escherichia coli
: Involvement of
alsI
and Regulation of
als
Regulon Expression by Allose and Ribose
Authors: Poulsen Tim S. , Chang Ying-Ying , Hove-Jensen Bjarne
Published: December 2019

Genes involved in allose utilization of
Escherichia coli
K-12 are organized in at least two operons,
alsRBACE
and
alsI
, located next to each other on the chromosome but divergently transcribed. Mutants defective in
alsI
(allose 6-phosphate isomerase gene) and
alsE
(allulose 6-phosphate epimerase gene) were Als
−
. Transcription of the two allose operons, measured as β-galactosidase activity specified by
alsI-lacZ
+
or
alsE-lacZ
+
operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired.
Providencia stuartii
Genes Activated by Cell-to-Cell Signaling and Identification of a Gene Required for Production or Activity of an Extracellular Factor
Authors: Rather Philip N. , Ding Xuedong , Baca-DeLancey Rita R. , Siddiqui Soofia
Published: December 2019

By utilizing reporter transposons, five
Providencia stuartii
genes that are activated by the accumulation of self-produced extracellular signals have been identified. These genes have been designated
cma
for conditioned medium activated. The presence of conditioned medium from stationary-phase cultures grown in rich media resulted in the premature activation of each gene in cells at early log phase, with activation values ranging from 6- to 26-fold. Preparation of conditioned medium from an M9 salts medium and fractionation by gel filtration chromatography resulted in fractions within the included volume which activated three of the
cma
fusions. In addition, depending on the reporter fusion, peak activity was found in different fractions. The partially purified factors activated in a dose-dependent manner. Characterization of the factors activating the
cma
fusions indicated that they were stable to heat, alkali, and acid. Furthermore, for each
cma
fusion, factor activity was not reproduced by the addition of homoserine lactone, homocysteine thiolactone, pyruvate, Casamino Acids, or α-ketoglutarate. The identities of three
cma
genes have been determined and revealed physiological roles in amino acid biosynthesis and nutrient import. To begin to address the pathways for production of or response to the extracellular factors, we have identified a locus,
aarA
, that is required for the activation of four
cma
fusions. The AarA product was required for factor activity in extracellular supernatants, indicating a possible role in biosynthesis or export.
Unusual Structure of the
attB
Site of the Site-Specific Recombination System of
Lactobacillus delbrueckii
Bacteriophage mv4
Authors: Auvray Frédéric , Coddeville Michèle , Ordonez Romy Catoira , Ritzenthaler Paul
Published: December 2019

The temperate phage mv4 integrates its genome into the chromosome of
Lactobacillus delbrueckii
subsp.
bulgaricus
by site-specific recombination within the 3′ end of a tRNA
Ser
gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage
attP
site and the bacterial
attB
site. In this study, we show that the mv4 integrase functions in vivo in
Escherichia coli
and we characterize the bacterial
attB
site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the
attB
sequence was determined by site-directed mutagenesis. The structure of the
attB
site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA
Ser
gene were present within the minimal
attB
site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.
Localization of Synthesis of β1,6-Glucan in
Saccharomyces cerevisiae
Authors: Montijn Roy C. , Vink Edwin , Müller Wally H. , Verkleij Arie J. , Van Den Ende Herman , Henrissat Bernard , Klis Frans M.
Published: December 2019

β1,6-Glucan is a key component of the yeast cell wall, interconnecting cell wall proteins, β1,3-glucan, and chitin. It has been postulated that the synthesis of β1,6-glucan begins in the endoplasmic reticulum with the formation of protein-bound primer structures and that these primer structures are extended in the Golgi complex by two putative glucosyltransferases that are functionally redundant, Kre6 and Skn1. This is followed by maturation steps at the cell surface and by coupling to other cell wall macromolecules. We have reinvestigated the role of Kre6 and Skn1 in the biogenesis of β1,6-glucan. Using hydrophobic cluster analysis, we found that Kre6 and Skn1 show significant similarities to family 16 glycoside hydrolases but not to nucleotide diphospho-sugar glycosyltransferases, indicating that they are glucosyl hydrolases or transglucosylases instead of genuine glucosyltransferases. Next, using immunogold labeling, we tried to visualize intracellular β1,6-glucan in cryofixed
sec1-1
cells which had accumulated secretory vesicles at the restrictive temperature. No intracellular labeling was observed, but the cell surface was heavily labeled. Consistent with this, we could detect substantial amounts of β1,6-glucan in isolated plasma membrane-derived microsomes but not in post-Golgi secretory vesicles. Taken together, our data indicate that the synthesis of β1,6-glucan takes place largely at the cell surface. An alternative function for Kre6 and Skn1 is discussed.
The Genetic Switch Regulating Activity of Early Promoters of the Temperate Lactococcal Bacteriophage TP901-1
Authors: Madsen Peter Lynge , Johansen Annette H. , Hammer Karin , Brøndsted Lone
Published: December 2019

A functional analysis of open reading frame 4 (ORF4) and ORF5 from the temperate lactococcal phage TP901-1 was performed by mutant and deletion analysis combined with transcriptional studies of the early phage promoters
p
R
and
p
L
. ORF4 (180 amino acids) was identified as a phage repressor necessary for repression of both promoters. Furthermore, the presence of ORF4 confers immunity of the host strain to TP901-1. ORF5 (72 amino acids) was found to be able to inhibit repression of the lytic promoter
p
L
by ORF4. Upon transformation with a plasmid containing both ORF4 and ORF5 and their cognate promoters, clonal variation is observed: in each transformant, either
p
L
is open and
p
R
is closed or vice versa. The repression is still dependent on ORF4, and the presence of ORF5 is needed for the clonal variation. Induction of a repressed
p
L
fusion containing
orf4
and
orf5
was obtained by addition of mitomycin C, and the induction was also shown to be dependent on the presence of the RecA protein, even though ORF4 does not contain a recognizable autocleavage site. Our results suggest that the relative amounts of the two proteins ORF4 and ORF5 determine the decision between lytic or lysogenic life cycle after phage infection and that a protein complex consisting of ORF4 and ORF5 may constitute a new type of genetic switch in bacteriophages.
A Multifunctional ATP-Binding Cassette Transporter System from
Vibrio cholerae
Transports Vibriobactin and Enterobactin
Authors: Wyckoff Elizabeth E. , Valle Ana-Maria , Smith Stacey L. , Payne Shelley M.
Published: December 2019

Vibrio cholerae
uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes,
viuP
,
viuD
,
viuG
and
viuC
, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [
3
H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in
Escherichia coli
. In the same assay, the
E. coli
enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire
viuPDGC
system could complement mutations in
fepB
,
fepD
,
fepG
, or
fepC
, only
viuC
was able to independently complement the corresponding
fep
mutation. This indicates that these proteins usually function as a complex.
V. cholerae
strains carrying a mutation in
viuP
or in
viuG
were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that
V. cholerae
contains genes in addition to
viuPDGC
that function in the transport of catechol siderophores.
Promiscuous Origin of a Chimeric Sequence in the
Escherichia coli
O157:H7 Genome
Authors: LeClerc J. Eugene , Li Baoguang , Payne William L. , Cebula Thomas A.
Published: December 2019

A novel sequence of 2.9 kb in the intergenic region between the
mutS
and
rpoS
genes of
Escherichia coli
O157:H7 and closely related strains replaces a sequence of 6.1 kb in
E. coli
K-12 strains. At the same locus in
Shigella dysenteriae
type 1, a sequence identical to that in O157:H7 is bounded by the IS
1
insertion sequence element. Extensive polymorphism in the
mutS-rpoS
chromosomal region is indicative of horizontal transfer events.
Spent Culture Supernatant of
Mycobacterium tuberculosis
H37Ra Improves Viability of Aged Cultures of This Strain and Allows Small Inocula To Initiate Growth
Authors: Sun Zhonghe , Zhang Ying
Published: December 2019

Spent culture supernatant from early-stationary-phase
Mycobacterium tuberculosis
H37Ra cultures increased the viability of bacilli from aged cultures of this strain and allowed small inocula to initiate growth in liquid culture. The resuscitation factor was acid labile and heat stable, with a mass of less than 1,375 Da.
IS
6110
-Mediated Deletions of Wild-Type Chromosomes of
Mycobacterium tuberculosis
Authors: Fang Z. , Doig C. , Kenna D. T. , Smittipat N. , Palittapongarnpim P. , Watt B. , Forbes K. J.
Published: December 2019

The
ipl
locus is a site for the preferential insertion of IS
6110
and has been identified as an insertion sequence, IS
1547
, in its own right. Various deletions around the
ipl
locus of clinical isolates of
Mycobacterium tuberculosis
were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS
6110
copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS
6110
transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS
6110
was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of
M. tuberculosis
, for IS
6110
-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of
M. tuberculosis
has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.
The
udhA
Gene of
Escherichia coli
Encodes a Soluble Pyridine Nucleotide Transhydrogenase
Authors: Boonstra Birgitte , French Christopher E. , Wainwright Ian , Bruce Neil C.
Published: December 2019

The
udhA
gene of
Escherichia coli
was cloned and expressed in
E. coli
and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published
E. coli
genome sequences due to a sequencing error.
E. coli
is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.
Analysis of FtsZ Assembly by Light Scattering and Determination of the Role of Divalent Metal Cations
Authors: Mukherjee Amit , Lutkenhaus Joe
Published: December 2019

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90° angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg
2+
was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca
2+
mediated dynamic assembly of FtsZ (X.-C. Yu and W. Margolin, EMBO J. 16:5455–5463, 1997). However, we found that Ca
2+
was not required for FtsZ assembly and that this concentration of Ca
2+
reduced the dynamic behavior of FtsZ assembly.
The SPI-3 Pathogenicity Island of
Salmonella enterica
Authors: Blanc-Potard Anne-Béatrice , Solomon Felix , Kayser Jayson , Groisman Eduardo A.
Published: December 2019

Pathogenicity islands are chromosomal clusters of pathogen-specific virulence genes often found at tRNA loci. We have determined the molecular genetic structure of SPI-3, a 17-kb pathogenicity island located at the
selC
tRNA locus of
Salmonella enterica
serovar Typhimurium. The G+C content of SPI-3 (47.5%) differs from that of the
Salmonella
genome (52%), consistent with the notion that these sequences have been horizontally acquired. SPI-3 harbors 10 open reading frames organized in six transcriptional units, which include the previously described
mgtCB
operon encoding the macrophage survival protein MgtC and the Mg
2+
transporter MgtB. Among the newly identified open reading frames, one exhibits sequence similarity to the ToxR regulatory protein of
Vibrio cholerae
and one is similar to the AIDA-I adhesin of enteropathogenic
Escherichia coli
. The distribution of SPI-3 sequences varies among the salmonellae: the right end of the island, which harbors the virulence gene
mgtC
, is present in all eight subspecies of
Salmonella
; however, a four-gene cluster at the center of SPI-3 is found in only some of the subspecies and is bracketed by remnants of insertion sequences, suggesting a multistep process in the evolution of SPI-3 sequences.
Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids
Authors: Takahashi Shunji , Kuzuyama Tomohisa , Seto Haruo
Published: December 2019

The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC
1.1.1.34
) was purified 3,000-fold from
Streptomyces
sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH
4
)
2
SO
4
precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent

K
m

values of 62 μM for NADPH and 7.7 μM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.
Cloning and Characterization of Arylamine N -Acetyltransferase Genes from
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
: Increased Expression Results in Isoniazid Resistance
Authors: Payton Mark , Auty Roy , Delgoda Rupika , Everett Martin , Sim Edith
Published: December 2019

Arylamine
N
-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote
Salmonella typhimurium
. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it.
nat
genes were cloned from
Mycobacterium smegmatis
and
Mycobacterium tuberculosis
, and expressed in
Escherichia coli
and
M. smegmatis
. The induced
M. smegmatis
NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from
S. typhimurium
recognizes NAT from
M. smegmatis
and cross-reacts with recombinant NAT from
M. tuberculosis
. Overexpression of mycobacterial
nat
genes in
E. coli
results in predominantly insoluble recombinant protein; however, with
M. smegmatis
as the host using the vector pACE-1, NAT proteins from
M. tuberculosis
and
M. smegmatis
are soluble.
M. smegmatis
transformants induced to express the
M. tuberculosis nat
gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.
Chromosomal Changes during Experimental Evolution in Laboratory Populations of
Escherichia coli
Authors: Bergthorsson Ulfar , Ochman Howard
Published: December 2019

Short-term rates of chromosome evolution were analyzed in experimental populations of
Escherichia coli
B that had been propagated for 2,000 generations under four thermal regimens. Chromosome alterations were monitored in 24 independent populations by pulsed-field gel electrophoresis of DNA treated with five rare-cutting restriction enzymes. A total of 11 changes, 8 affecting chromosome size and 3 altering restriction sites, were observed in these populations, with none occurring in strains cultured at 37°C. Considering the changes detected in these experimental populations, the rate of chromosome alteration of
E. coli
is estimated to be half of that observed in experimental populations of yeast.
Lack of Genetic Differentiation between Two Geographically Diverse Samples of
Candida albicans
Isolated from Patients Infected with Human Immunodeficiency Virus
Authors: Xu Jianping , Vilgalys Rytas , Mitchell Thomas G.
Published: December 2019

The patterns of genetic variation of samples of
Candida albicans
isolated from patients infected with human immunodeficiency virus in Durham, N.C., and Vitória, Brazil, were compared. Genotypes for 126 strains were obtained at 16 polymorphic restriction sites distributed on nine PCR fragments. The results indicated evidence of clonality both within and between these two geographically diverse samples. The samples are genetically very similar, with little evidence of genetic differentiation.
Ferritin Mutants of
Escherichia coli
Are Iron Deficient and Growth Impaired, and
fur
Mutants are Iron Deficient
Authors: Abdul-Tehrani Hossein , Hudson Aaron J. , Chang Yung-Sheng , Timms Andrew R. , Hawkins Chris , Williams John M. , Harrison Pauline M. , Guest John R. , Andrews Simon C.
Published: December 2019

Escherichia coli
contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (
ftnA
and
bfr
) were inactivated by replacing the chromosomal
ftnA
and
bfr
genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (
ftnA::spc
and
bfr::kan
) and a double mutant (
ftnA::spc bfr::kan
) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the
bfr
mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the
ftnA
and
ftnA bfr
mutants remained unchanged. The
ftnA
and
ftnA bfr
mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (
fur
) mutants also had very low iron contents (2.5-fold less iron than Fur
+
strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the
ftnA
and
fur
mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of
ftnA
mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the
fur
mutation,
ftnA
and
bfr
mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.