bluuebunny/crossref_metadata_2025 huggingface dataset:
Campylobacter fetus
Surface Layer Proteins Are Transported by a Type I Secretion System
Authors: Thompson Stuart A. , Shedd Omer L. , Ray Kevin C. , Beins Michael H. , Jorgensen Jesse P. , Blaser Martin J.
Published: December 2019

The virulence of
Campylobacter fetus
, a bacterial pathogen of ungulates and humans, is mediated in part by the presence of a paracrystalline surface layer (S-layer) that confers serum resistance. The subunits of the S-layer are S-layer proteins (SLPs) that are secreted in the absence of an N-terminal signal sequence and attach to either type A or B
C. fetus
lipopolysaccharide in a serospecific manner. Antigenic variation of multiple SLPs (encoded by
sapA
homologs) of type A strain 23D occurs by inversion of a promoter-containing DNA element flanked by two
sapA
homologs. Cloning and sequencing of the entire 6.2-kb invertible region from
C. fetus
23D revealed a probable 5.6-kb operon of four overlapping genes (
sapCDEF
, with sizes of 1,035, 1,752, 1,284, and 1,302 bp, respectively) transcribed in the opposite direction from
sapA
. The four genes also were present in the invertible region of type B strain 84-107 and were virtually identical to their counterparts in the type A strain. Although SapC had no database homologies, SapD, SapE, and SapF had predicted amino acid homologies with type I protein secretion systems (typified by
Escherichia coli
HlyBD/TolC or
Erwinia chrysanthemi
PrtDEF) that utilize C-terminal secretion signals to mediate the secretion of hemolysins, leukotoxins, or proteases from other bacterial species. Analysis of the C termini of four
C. fetus
SLPs revealed conserved structures that are potential secretion signals. A
C. fetus sapD
mutant neither produced nor secreted SLPs.
E. coli
expressing
C. fetus sapA
and
sapCDEF
secreted SapA, indicating that the
sapCDEF
genes are sufficient for SLP secretion.
C. fetus
SLPs therefore are transported to the cell surface by a type I secretion system.
The EII
Glc
Protein Is Involved in Glucose-Mediated Activation of
Escherichia coli gapA
and
gapB-pgk
Transcription
Authors: Charpentier B. , Bardey V. , Robas N. , Branlant C.
Published: December 2019

The
Escherichia coli gapB
gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH). In most bacteria, the gene for GAPDH is located upstream of the
pgk
gene encoding 3-phosphoglycerate kinase (PGK). This is the case for
gapB
. However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH). The active GAPDH is encoded by the
gapA
gene. Since we found that the nucleotide region upstream of the
gapB
open reading frame is responsible for part of the PGK production, we analyzed
gapB
promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription. We showed the presence of a unique transcription promoter,
gapB
P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site. Interestingly, the
gapB
P0 promoter activity was strongly enhanced when glucose was used as the carbon source. In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter
gapB
P0 activity. In contrast, abolition of CRP production or of cAMP biosynthesis (
crp
or
cya
mutant strains) strongly reduced promoter
gapB
P0 activity. This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter
gapB
P0 activity. We also showed that glucose stimulation of
gapB
P0 promoter activity depends on the expression of enzyme II
Glc
(EII
Glc
), encoded by the
ptsG
gene, and that the
gapA
P1 promoter is also activated by glucose via the EII
Glc
protein. A similar glucose-mediated activation, dependent on the EII
Glc
protein, was described by others for the
pts
operon. Altogether, this shows that when glucose is present in the growth medium expression of the
E. coli
genes required for its uptake (
pts
) and its metabolism (
gapA
and
gapB-pgk
) are coordinately activated by a mechanism dependent upon the EII
Glc
protein.
Region of Flo1 Proteins Responsible for Sugar Recognition
Authors: Kobayashi Osamu , Hayashi Nobuyuki , Kuroki Ryota , Sone Hidetaka
Published: December 2019

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The
FLO1
gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of
FLO1
, named Lg-
FLO1
, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both
FLO1
and Lg-
FLO1
are involved in the yeast flocculation phenomenon, the
FLO1
gene in the mannose-specific flocculation strain was replaced by the Lg-
FLO1
gene. The transformant in which the Lg-
FLO1
gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the
FLO1
and Lg-
FLO1
genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric
FLO1
and Lg-
FLO1
genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.
A MADS Box Protein Consensus Binding Site Is Necessary and Sufficient for Activation of the Opaque-Phase-Specific Gene
OP4
of
Candida albicans
Authors: Lockhart Shawn R. , Nguyen Mau , Srikantha Thyagarajan , Soll David R.
Published: December 2019

The majority of strains of
Candida albicans
can switch frequently and reversibly between two or more general phenotypes, a process now considered a putative virulence factor in this species.
Candida albicans
WO-1 switches frequently and reversibly between a white and an opaque phase, and this phenotypic transition is accompanied by the differential expression of white-phase-specific and opaque-phase-specific genes. In the opaque phase, cells differentially express the gene
OP4
, which encodes a putative protein 402 amino acids in length that contains a highly hydrophobic amino-terminal sequence and a carboxy-terminal sequence with a pI of 10.73. A series of deletion constructs fused to the
Renilla reniformis
luciferase was used to functionally characterize the
OP4
promoter in order to investigate how this gene is differentially expressed in the white-opaque transition. An extremely strong 17-bp transcription activation sequence was identified between −422 and −404 bp. This sequence contained a MADS box consensus binding site, most closely related to the Mcm1 binding site of
Saccharomyces cerevisiae
. A number of point mutations generated in the MADS box consensus binding site as well as a complete deletion of the consensus site further demonstrated that it was essential for the activation of
OP4
transcription in the opaque phase. Gel mobility shift assays with the 17-bp activation sequence identified three specific complexes which formed with both white- and opaque-phase cell extracts. Competition with a putative MADS box consensus binding site from the promoter of the coordinately regulated opaque-phase-specific gene
PEP1 (SAP1)
and the human MADS box consensus binding site for serum response factor demonstrated that one of the three complexes formed was specific to the
OP4
sequence.
The Hsc66-Hsc20 Chaperone System in
Escherichia coli
: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System
Authors: Silberg Jonathan J. , Hoff Kevin G. , Vickery Larry E.
Published: December 2019

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in
Escherichia coli
. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.
Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of
Escherichia coli
Authors: Benoit Stéphane , Abaibou Hafid , Mandrand-Berthelot Marie-Andrée
Published: December 2019

Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate,
Escherichia coli
synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using
blaM
(β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome
b
, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the
35
S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.
Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions
Authors: He Luhong , Soupene Eric , Ninfa Alexander , Kustu Sydney
Published: December 2019

In
Klebsiella pneumoniae
, NifA-dependent transcription of nitrogen fixation (
nif
) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for
glnK
, which encodes a P
II
-like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria—apparently a paralogue of GlnK—cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the
glnD
gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnK
Y51N
, which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.
Coupled Changes in Translation and Transcription during Cobalamin-Dependent Regulation of
btuB
Expression in
Escherichia coli
Authors: Nou Xiangwu , Kadner Robert J.
Published: December 2019

The level of the vitamin B
12
transport protein BtuB in the outer membrane of
Escherichia coli
is strongly reduced by growth in the presence of cobalamins. Previous analyses of regulatory mutants and of
btuB-lacZ
fusions indicated that the primary site of
btuB
gene regulation was at the translational level, and this required sequences throughout the 240-nucleotide (nt) leader region. Cobalamin-dependent regulation of transcriptional fusions was of a lesser magnitude but required, in addition to the leader, sequences within the first 100 nt of the coding sequence, termed the translated regulatory region (TRR). To analyze the process of transcription-level regulation of
btuB
in
E. coli
, the levels and metabolism of
btuB
RNA were analyzed by S1 nuclease protection assays, and mutations that alter the coupling of translational and transcriptional control were analyzed. Expression of transcriptional fusions was found to correlate with changes in the level of intact
btuB
RNA and was related to changes in the metabolic stability of the normally long-lived RNA. Mutational analysis showed that the
btuB
start codon and a hairpin structure that can sequester the Shine-Dalgarno sequence are necessary for cobalamin-dependent regulation and that translation of the TRR is necessary for extended RNA stability and for expression of the transcriptional fusion. The absence of regulation at the stage of transcription initiation was confirmed by the findings that several truncated
btuB
RNA fragments were expressed in a constitutive manner and that the normal regulatory response occurred even when the
btuB
promoter and upstream sequences were replaced by the heterologous
bla
and
lac
promoters. Transcription driven by phage T7 RNA polymerase was not regulated by cobalamins, although some regulation at the translational level was retained. Cobalamin-dependent changes in RNA structure were suggested from the RNase III-dependent production of a transcript fragment that is made only in the presence of cobalamin and is independent of the regulatory outcome. These results indicate that the primary control of
btuB
expression by cobalamin occurs at the level of translation initiation, which directly affects the level and stability of
btuB
RNA in a process that requires the presence of the intact translated regulatory region.
Cloning and Expression of the Gene for the Na
+
-Coupled Serine Transporter from
Escherichia coli
and Characteristics of the Transporter
Authors: Ogawa Wakano , Kim Young-Mog , Mizushima Tohru , Tsuchiya Tomofusa
Published: December 2019

We cloned a gene (
sstT
) for the Na
+
/serine symporter from the chromosome of
Escherichia coli
by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.
The
aadA
Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in
Myxococcus xanthus
Authors: Magrini Vincent , Creighton Chad , White David , Hartzell Patricia L. , Youderian Philip
Published: December 2019

Plasmids with the
aadA
gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in
Escherchia coli
, can be introduced into wild-type
Myxococcus xanthus
, strain DK1622, by electroporation. Recombinant
M. xanthus
strains with integrated plasmids carrying the
aadA
gene acquire resistance to high levels of these antibiotics. Selection for
aadA
in
M. xanthus
can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with
aadA
and the
M. xanthus
chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the
M. xanthus
genome favors the targeted disruption of
M. xanthus
genes by allele replacement.
Molecular Mechanism of Peptide-Specific Pheromone Signaling in
Enterococcus faecalis
: Functions of Pheromone Receptor TraA and Pheromone-Binding Protein TraC Encoded by Plasmid pPD1
Authors: Nakayama Jiro , Takanami Yuuichiro , Horii Takaaki , Sakuda Shohei , Suzuki Akinori
Published: December 2019

Conjugative transfer of the
Enterococcus faecalis
plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [
3
H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [
3
H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [
3
H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the
traA
-bearing multicopy plasmid (pDLHH21) also exhibited high [
3
H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 ± 0.08 nM against [
3
H]cPD1. iPD1 competitively inhibited [
3
H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the
traC
-bearing multicopy plasmid (pDLES23) exhibited significant [
3
H]cPD1-binding activity. A strain carrying
traC
-disrupted pPD1 (pAM351CM) exhibited lower [
3
H]cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [
3
H]cPD1 binding to the
traC
transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.
Identification and Characterization of Protease-Resistant SecA Fragments: SecA Has Two Membrane-Integral Forms
Authors: Chen Xianchuan , Brown Timothy , Tai Phang C.
Published: December 2019

We have identified and characterized the protease-resistant SecA fragments (X. Chen, H. Xu, and P. C. Tai, J. Biol. Chem. 271:29698–29706, 1996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis. The 66-, 36-, and 27-kDa proteolytic fragments in the membranes all start at Met
1
, whereas the 48-kDa fragment starts at Glu
361
. The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from different SecA molecules. These two fragments were generated differently in response to ATP hydrolysis and protein translocation. Furthermore, the presence of membrane is required for the generation of the 48-kDa fragment but not for that of the 66-kDa fragment. These data suggest that there are two different integral forms of SecA in the membrane: SecA
S
and SecA
M
. The combination of these two forms of SecA has several membrane-interacting domains. Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa fragments could be derived from urea- or Na
2
CO
3
-washed membranes. Moreover, all fragments are resistant to extraction with a high concentration of salt or with heparin, but the membrane-specific 48-kDa SecA domain is more sensitive to Na
2
CO
3
or urea extraction. This suggests that this domain may interact with other membrane proteins in an aqueous microenvironment and therefore may form a part of the protein-conducting channel.
The Yersiniabactin Biosynthetic Gene Cluster of
Yersinia enterocolitica
: Organization and Siderophore-Dependent Regulation
Authors: Pelludat C. , Rakin A. , Jacobi C. A. , Schubert S. , Heesemann J.
Published: December 2019

The ability to synthesize and uptake the
Yersinia
siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species
Yersinia pestis
,
Y. pseudotuberculosis
, and
Y. enterocolitica
1B. We have identified four genes,
irp1
,
irp3
,
irp4
, and
irp5
, on a 13-kb chromosomal DNA fragment of
Y. enterocolitica
O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined
irp2
. The
irp1
gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of
irp1
show similarity to the corresponding regions of the polyketide synthase genes of
Bacillus subtilis
and
Streptomyces antibioticus
. The remaining part of
irp1
is most similar to
irp2
, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of
Vibrio anguillarum
. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of
Escherichia coli
. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in
Y. pestis. irp3
has no similarity to any known gene. Inactivation of either
irp1
or
irp2
abrogates yersiniabactin synthesis. Mutations in
irp1
or
fyuA
(encoding yersiniabactin/pesticin receptor) result in downregulation of
irp2
that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an
irp1
mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.
Alanine Catabolism in
Klebsiella aerogenes
: Molecular Characterization of the
dadAB
Operon and Its Regulation by the Nitrogen Assimilation Control Protein
Authors: Janes Brian K. , Bender Robert A.
Published: December 2019

Klebsiella aerogenes
strains with reduced levels of
d
-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in
dadA
(the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of
Klebsiella aerogenes
,
dadAB
, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of
dadA
strains. The operon has a high similarity to the
dadAB
operon of
Salmonella typhimurium
and the
dadAX
operon of
Escherichia coli
, each of which codes for the smaller subunit of
d
-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for
E. coli
and
S. typhimurium
, the
dad
operon of
K. aerogenes
is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position −44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.
Assignment of Biochemical Functions to Glycosyl Transferase Genes Which Are Essential for Biosynthesis of Exopolysaccharides in
Sphingomonas
Strain S88 and
Rhizobium leguminosarum
Authors: Pollock Thomas J. , van Workum Wilbert A. T. , Thorne Linda , Mikolajczak Marcia J. , Yamazaki Motohide , Kijne Jan W. , Armentrout Richard W.
Published: December 2019

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The
spsK
gene of
Sphingomonas
strain S88 and the
pssDE
genes of
Rhizobium leguminosarum
were identified as encoding glucuronosyl-(β1→4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the
spsK
gene and the
pssDE
genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that
spsL
codes for a glucosyl-(β1→4)-glucuronosyl transferase in
Sphingomonas
and that
pssC
codes for a glucuronosyl-(β1→4)-glucuronosyl transferase in
R. leguminosarum
. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C
55
-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.
Modulation of the Function of the Signal Receptor Domain of XylR, a Member of a Family of Prokaryotic Enhancer-Like Positive Regulators
Authors: Salto Rafael , Delgado Asunción , Michán Carmen , Marqués Silvia , Ramos Juan L.
Published: December 2019

The XylR protein controls expression from the
Pseudomonas putida
TOL plasmid upper pathway operon promoter (Pu) in response to aromatic effectors. XylR-dependent stimulation of transcription from a Pu::
lacZ
fusion shows different induction kinetics with different effectors. With toluene, activation followed a hyperbolic curve with an apparent
K
of 0.95 mM and a maximum β-galactosidase activity of 2,550 Miller units. With
o
-nitrotoluene, in contrast, activation followed a sigmoidal curve with an apparent
K
of 0.55 mM and a Hill coefficient of 2.65.
m
-Nitrotoluene kept the XylR regulator in an inactive transcriptional form. Therefore, upon binding of an effector, the substituent on the aromatic ring leads to productive or unproductive XylR forms. The different transcriptional states of the XylR regulator are substantiated by XylR mutants. XylRE172K is a mutant regulator that is able to stimulate transcription from the Pu promoter in the presence of
m
-nitrotoluene; however, its response to
m
-aminotoluene was negligible, in contrast with the wild-type regulator. These results illustrate the importance of the electrostatic interactions in effector recognition and in the stabilization of productive and unproductive forms by the regulator upon aromatic binding. XylRD135N and XylRD135Q are mutant regulators that are able to stimulate transcription from Pu in the absence of effectors, whereas substitution of Glu for Asp135 in XylRD135E resulted in a mutant whose ability to recognize effectors was severely impaired. Therefore, the conformation of mutant XylRD135Q as well as XylRD135N seemed to mimic that of the wild-type regulator when effector binding occurred, whereas mutant XylRD135E seemed to be blocked in a conformation similar to that of wild-type XylR and XylRE172K upon binding to an inhibitor molecule such as
m
-nitrotoluene or
m
-aminotoluene.
std1
, a Gene Involved in Glucose Transport in
Schizosaccharomyces pombe
Authors: Mehta Shwetal V. , Patil Vandana B. , Velmurugan S. , Lobo Zita , Maitra Pabitra K.
Published: December 2019

A wild-type strain, Sp972 h
−
, of
Schizosaccharomyces pombe
was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as
std1-8
and
std1-23
(sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [
14
C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in
S. pombe
, unlike in
Saccharomyces cerevisiae
. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.
Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the
n
-Alkane-Assimilating Yeast
Candida tropicalis
Authors: Kanayama Naoki , Ueda Mitsuyoshi , Atomi Haruyuki , Tanaka Atsuo
Published: December 2019

The
n
-alkane-assimilating diploid yeast
Candida tropicalis
possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by
CT-T1A
and
CT-T1B
, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by
CT-T3A
and
CT-T3B
. The physiological functions of these thiolases have been examined by gene disruption. The homozygous
ct-t1aΔ/t1b
Δ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous
ct-t3aΔ/t3b
Δ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on
n
-alkanes (C
10
to C
13
). All thiolase activities in this yeast disappeared with the
ct-t1aΔ/t1b
Δ and
ct-t3aΔ/t3b
Δ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the
CT-T1A
locus in the
ct-t1b
Δ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C
4
substrate butyrate.
Intragenic Suppressors of Induction-Deficient TetR Mutants: Localization and Potential Mechanism of Action
Authors: Biburger Markus , Berens Christian , Lederer Thomas , Krec Traudl , Hillen Wolfgang
Published: December 2019

Eight Tn
10
Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline. The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor. Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction.
Two Separate Regulatory Systems Participate in Control of Swarming Motility of
Serratia liquefaciens
MG1
Authors: Givskov Michael , Östling Jörgen , Eberl Leo , Lindum Peter W. , Christensen Allan Beck , Christiansen Gunna , Molin Søren , Kjelleberg Staffan
Published: December 2019

Swarming motility of
Serratia liquefaciens
MG1 requires the expression of two genetic loci,
flhDC
and
swrI
. Here we demonstrate that the products of the
flhDC
operon (the flagellar master regulator) and the
swrI
gene (the extracellular signal molecule
N
-butanoyl-
l
-homoserine lactone) are global regulators which control two separate regulons.
Contact Stimulation of Tgl and Type IV Pili in
Myxococcus xanthus
Authors: Wall Daniel , Wu Samuel S. , Kaiser Dale
Published: December 2019

Myxococcus xanthus tgl
mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting
tgl
+
cells. The absence of pili in
tgl
mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.
A Functional Homolog of
Escherichia coli
NhaR in
Vibrio cholerae
Authors: Williams S. G. , Carmel-Harel O. , Manning P. A.
Published: December 2019

Escherichia coli
NhaR controls expression of a sodium/proton (Na
+
/H
+
) antiporter, NhaA. The
Vibrio cholerae
NhaR protein shows over 60% identity to those of
Escherichia coli
and
Salmonella enteritidis. V. cholerae
NhaR complements an
E. coli nhaR
mutant for growth in 100 mM LiCl–33 mM NaCl, pH 7.6, and enhances the Na
+
-dependent induction of an
E. coli
chromosomal
nhaA
::
lacZ
fusion. These findings indicate functional homology to
E. coli
NhaR. Two
V. cholerae nhaR
mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of
V. cholerae nhaR
on a low-copy-number plasmid. An
nhaR
mutation had no detectable effect on the virulence of the
V. cholerae
strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.
Mutational Biosynthesis of Novel Rapamycins by a Strain of
Streptomyces hygroscopicus
NRRL 5491 Disrupted in
rapL
, Encoding a Putative Lysine Cyclodeaminase
Authors: Khaw Lake Ee , Böhm Günter A. , Metcalfe Su , Staunton James , Leadlay Peter F.
Published: December 2019

The gene
rapL
lies within the region of the
Streptomyces hygroscopicus
chromosome which contains the biosynthetic gene cluster for the immunosuppressant rapamycin. Introduction of a frameshift mutation into
rapL
by ΦC31 phage-mediated gene replacement gave rise to a mutant which did not produce significant amounts of rapamycin. Growth of this
rapL
mutant on media containing added
l
-pipecolate restored wild-type levels of rapamycin production, consistent with a proposal that
rapL
encodes a specific
l
-lysine cyclodeaminase important for the production of the
l
-pipecolate precursor. In the presence of added proline derivatives,
rapL
mutants synthesized novel rapamycin analogs, indicating a relaxed substrate specificity for the enzyme catalyzing pipecolate incorporation into the macrocycle.
tRNA
Arg
(
fimU
) and Expression of SEF14 and SEF21 in
Salmonella enteritidis
Authors: Clouthier S. C. , Collinson S. K. , White A. P. , Banser P. A. , Kay W. W.
Published: December 2019

A Tn
10
insertion affecting SEF14 fimbrial synthesis in
Salmonella enteritidis
was located 13 bp upstream of a gene designated
fimU
. The 77-bp DNA sequence of
fimU
from
S. enteritidis
was identical to that of
fimU
encoding tRNA
Arg
(UCU) from
Salmonella typhimurium
and 96% identical to that of the
Escherichia coli argU
homolog. Furthermore, the open reading frame adjacent to and overlapping the 3′ end of
fimU
was similar to the prophage DLP12 integrase gene. The
fimU
-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU. Thus,
fimU
encoded a tRNA
Arg
specific for the rare codon AGA.
fimU
mapped to the SEF21
fim
operon located 15 C’s from the
sef14
gene cluster. Although
fimU
was located within the SEF21
fim
gene cluster, the
fimU
Tn
10
insertion mutant of
S. enteritidis
was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production. SEF17 and SEF18 fimbria production was not affected. Complementation of this mutant with plasmid-borne
fimU
restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21. This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae.
Roles of DnaK and RpoS in Starvation-Induced Thermotolerance of
Escherichia coli
Authors: Rockabrand David , Livers Kevin , Austin Tess , Kaiser Robyn , Jensen Debra , Burgess Richard , Blum Paul
Published: December 2019

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in
Escherichia coli
. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these
dnaK
mutant phenotypes closely resemble those of
rpoS
(ς
38
) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the
dnaK
mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς
32
) suppressors of the
dnaK
mutant with restored levels of RpoS and
dnaK rpoS
double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved
E. coli.
Identification and Characterization of
alcR
, a Gene Encoding an AraC-Like Regulator of Alcaligin Siderophore Biosynthesis and Transport in
Bordetella pertussis
and
Bordetella bronchiseptica
Authors: Beaumont Fiona C. , Kang Ho Young , Brickman Timothy J. , Armstrong Sandra K.
Published: December 2019

A
Bordetella bronchiseptica
iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb
Kpn
I-
Pst
I
Bordetella pertussis
DNA fragment mapping downstream of the alcaligin biosynthesis genes
alcABC
restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation
alcR
. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the
alcR
open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an
Escherichia coli
expression system and visualized in electrophoretic gels. In-frame
alcR
deletion mutants of
B. pertussis
and
B. bronchiseptica
were constructed, and the defined mutants exhibited the
alcR
mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned
alcR
gene provided in
trans
restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of
Bordetella
alcaligin biosynthesis and transport genes and is required for their full expression.
Phosphorylation-Independent Activity of the Response Regulators AlgB and AlgR in Promoting Alginate Biosynthesis in Mucoid
Pseudomonas aeruginosa
Authors: Ma Sheng , Selvaraj Uma , Ohman Dennis E. , Quarless Ryan , Hassett Daniel J. , Wozniak Daniel J.
Published: December 2019

Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for
Pseudomonas aeruginosa
in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently,
kinB
was identified downstream of
algB
and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in
kinB
in a mucoid cystic fibrosis isolate,
P. aeruginosa
FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgBΔ1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgBΔ1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni
2+
affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgBΔ1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant
algB
alleles onto the chromosome of a
P. aeruginosa algB
mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation,
P. aeruginosa
strains expressing AlgB.D59N or H-AlgBΔ1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid
P. aeruginosa
appears not to be mediated by conventional phosphorylation-dependent signal transduction.
Cloning and Characterization of Transcription of the
xylAB
Operon in
Thermoanaerobacter ethanolicus
Authors: Erbeznik Milutin , Dawson Karl A. , Strobel Herbert J.
Published: December 2019

The genes encoding xylose isomerase (
xylA
) and xylulose kinase (
xylB
) from the thermophilic anaerobe
Thermoanaerobacter ethanolicus
were found to constitute an operon with the transcription initiation site 169 nucleotides upstream from the previously assigned (K. Dekker, H. Yamagata, K. Sakaguchi, and S. Udaka, Agric. Biol. Chem. 55:221–227, 1991) promoter region. The bicistronic
xylAB
mRNA was processed by cleavage within the 5′-terminal portion of the XylB-coding sequence. Transcription of
xylAB
was induced in the presence of xylose, and, unlike in all other xylose-utilizing bacteria studied, was not repressed by glucose. The existence of putative
xyl
operator sequences suggested that xylose utilization is controlled by a repressor-operator mechanism. The
T. ethanolicus xylB
gene coded for a 500-amino-acid-residue protein with a deduced amino acid sequence highly homologous to those of other XylBs. This is the first report of an
xylB
nucleotide sequence and an
xylAB
operon from a thermophilic anaerobic bacterium.
Assembly of Both the Head and Tail of Bacteriophage Mu Is Blocked in
Escherichia coli groEL
and
groES
Mutants
Authors: Grimaud Régis , Toussaint Ariane
Published: December 2019

Like several other
Escherichia coli
bacteriophages, transposable phage Mu does not develop normally in
groE
hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol. 169:5504–5509, 1987). We show here that lysates obtained upon induction of
groE
Mu lysogens contain free inactive tails and empty heads. GroEL and GroES are thus essential for the correct assembly of both Mu heads and Mu tails. Evidence is presented that
groE
mutations inhibit processing of the phage head protein gpH as well as the formation of a 25S complex suspected to be an early Mu head assembly intermediate.
Salmonella typhimurium
Encodes an SdiA Homolog, a Putative Quorum Sensor of the LuxR Family, That Regulates Genes on the Virulence Plasmid
Authors: Ahmer Brian M. M. , van Reeuwijk Jeroen , Timmers Cynthia D. , Valentine Peter J. , Heffron Fred
Published: December 2019

Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones. In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression. The gene encoding one LuxR family member, named
sdiA
(suppressor of cell division inhibition), is present in the
Escherichia coli
genome. In this report, we have cloned the
Salmonella typhimurium
homolog of SdiA and performed a systematic screen for
sdiA
-regulated genes. A 4.4-kb fragment encoding the
S. typhimurium sdiA
gene was sequenced and found to encode the 3′ end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (
Salmonella
invasion regulator), and the 5′ end of UvrC. This gene organization is conserved between
E. coli
and
S. typhimurium
. We determined that the
S. typhimurium sdiA
gene was able to weakly complement the
E. coli sdiA
gene for activation of
ftsQAZ
at promoter 2 and for suppression of filamentation caused by an
ftsZ
(Ts) allele. To better understand the function of
sdiA
in
S. typhimurium
, we screened 10,000 random
lacZY
transcriptional fusions (MudJ transposon mutations) for regulation by
sdiA
. Ten positively regulated fusions were isolated. Seven of the fusions were within an apparent operon containing ORF8, ORF9,
rck
(resistance to complement killing), and ORF11 of the
S. typhimurium
virulence plasmid. The three ORFs have now been named
srgA
,
srgB
, and
srgC
(for
sdiA
-regulated gene), respectively. The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes.