bluuebunny/crossref_metadata_2025 huggingface dataset:
NasFED Proteins Mediate Assimilatory Nitrate and Nitrite Transport in
Klebsiella oxytoca
(
pneumoniae
) M5al
Authors: Wu Qitu , Stewart Valley
Published: December 2019

Klebsiella oxytoca
can use nitrate and nitrite as sole nitrogen sources. The enzymes required for nitrate and nitrite assimilation are encoded by the
nasFEDCBA
operon. We report here the complete
nasFED
sequence. Sequence comparisons indicate that the
nasFED
genes encode components of a conventional periplasmic binding protein-dependent transport system consisting of a periplasmic binding protein (NasF), a homodimeric intrinsic membrane protein (NasE), and a homodimeric ATP-binding cassette (ABC) protein (NasD). The NasF protein and the related NrtA and CmpA proteins of cyanobacteria contain leader (signal) sequences with the double-arginine motif that is hypothesized to direct prefolded proteins to an alternate protein export pathway. The NasE protein and the related NrtB and CmpB proteins of cyanobacteria contain unusual variants of the EAA loop sequence that defines membrane-intrinsic proteins of ABC transporters. To characterize nitrate and nitrite transport, we constructed in-frame nonpolar deletions of the chromosomal
nasFED
genes. Growth tests coupled with nitrate and nitrite uptake assays revealed that the
nasFED
genes are essential for nitrate transport and participate in nitrite transport as well. Interestingly, the Δ
nasF
strain exhibited leaky phenotypes, particularly at elevated nitrate concentrations, suggesting that the NasED proteins are not fully dependent on the NasF protein.
Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (
cwlJ
) Which Affects Germination of
Bacillus subtilis
Spores
Authors: Ishikawa Shu , Yamane Kunio , Sekiguchi Junichi
Published: December 2019

The predicted amino acid sequence of
Bacillus subtilis ycbQ
(renamed
cwlJ
) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of
B. cereus
and the deduced one of
B. subtilis
. We constructed a
cwlJ::lacZ
fusion in the
B. subtilis
chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the
cwlJ
gene indicated that it is transcribed by Eς
E
RNA polymerase.
cwlJ
-deficient spores responded to both
l
-alanine and AGFK, the
A
580
values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast,
B. subtilis sleB
spores did not release hexosamine at a significant level. While
cwlJ
and
sleB
spores were able to germinate, CJSB (
cwlJ sleB
) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in
A
580
and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of
sleB
mutant spores. The roles of the
sleB
and
cwlJ
mutations in germination and spore maturation are also discussed.
Helicobacter pylori ribBA
-Mediated Riboflavin Production Is Involved in Iron Acquisition
Authors: Worst Dennis J. , M. Gerrits Monique , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G.
Published: December 2019

In this study, we cloned and sequenced a DNA fragment from an ordered cosmid library of
Helicobacter pylori
NCTC 11638 which confers to a siderophore synthesis mutant of
Escherichia coli
(EB53
aroB hemA
) the ability to grow on iron-restrictive media and to reduce ferric iron. Sequence analysis of the DNA fragment revealed the presence of an open reading frame with high homology to the
ribA
gene of
Bacillus subtilis
. This gene encodes a bifunctional enzyme with the activities of both 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthase and GTP cyclohydrolase II, which catalyze two essential steps in riboflavin biosynthesis. Expression of the gene (designated
ribBA
) resulted in the formation of one translational product, which was able to complement both the
ribA
and the
ribB
mutation in
E. coli
. Expression of
ribBA
was iron regulated, as was suggested by the presence of a putative FUR box in its promotor region and as shown by RNA dot blot analysis. Furthermore, we showed that production of riboflavin in
H. pylori
cells is iron regulated.
E. coli
EB53 containing the plasmid with
H. pylori ribBA
excreted riboflavin in the culture medium, and this riboflavin excretion also appeared to be iron regulated. We postulate that the iron-regulated production of riboflavin and ferric-iron-reduction activity by
E. coli
EB53 transformed with the
H. pylori ribBA
gene is responsible for the survival of EB53 on iron-restrictive medium. Because disruption of
ribBA
in
H. pylori
eliminates its ferric-iron-reduction activity, we conclude that
ribBA
has an important role in ferric-iron reduction and iron acquisition by
H. pylori.
Role of the
fnrL
Gene in Photosystem Gene Expression and Photosynthetic Growth of
Rhodobacter sphaeroides
2.4.1
Authors: Zeilstra-Ryalls Jill H. , Kaplan Samuel
Published: December 2019

Anoxygenic photosynthetic growth of
Rhodobacter sphaeroides
2.4.1 requires a functional
fnrL
gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the
puc
operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in
puc
operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of
prr
or
ppsR
can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional
fnrL
gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode.
Xanthomonas campestris
pv. campestris
gum
Mutants: Effects on Xanthan Biosynthesis and Plant Virulence
Authors: Katzen Federico , Ferreiro Diego U. , Oddo Cristian G. , Ielmini M. Verónica , Becker Anke , Pühler Alfred , Ielpi Luis
Published: December 2019

Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium
Xanthomonas campestris
pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated
xpsI
or
gum
has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of
X. campestris gum
mutants, we report experimental data for assigning functions to the products of the
gum
genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain
gum
and non-
gum
mutations. In addition, we provide evidence that the C-terminal domain of the
gumD
gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of
X. campestris
against the plant.
Characterization of
dnaC2
and
dnaC28
Mutants by Flow Cytometry
Authors: Withers Helen L. , Bernander Rolf
Published: December 2019

Escherichia coli
strains containing thermosensitive
dnaC
alleles were studied by flow cytometry. Strains containing either the
dnaC2
or
dnaC28
allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of
dnaC
mutants for cell cycle studies is discussed.
Cloning and Characterization of the
Pseudomonas aeruginosa zwf
Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat)
Authors: Ma Ju-Fang , Hager Paul W. , Howell Michael L. , Phibbs Paul V. , Hassett Daniel J.
Published: December 2019

In this study, we cloned the
Pseudomonas aeruginosa zwf
gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD
+
- or NADP
+
-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predicted
zwf
gene product is 490 residues, which could form a tetramer with a molecular mass of ∼220 kDa. G6PDH activity and
zwf
transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and
zwf
transcription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be ∼220 kDa by size exclusion chromatography. Estimated

K
m

values of purified G6PDH acting on glucose-6-phosphate, NADP
+
, and NAD
+
were 530, 57, and 333 μM, respectively. The specific activities with NAD
+
and NADP
+
were calculated to be 176 and 69 μmol/min/mg. An isogenic
zwf
mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.
Genetic Complementation and Kinetic Analyses of
Rhodobacter capsulatus ORF1696
Mutants Indicate that the ORF1696 Protein Enhances Assembly of the Light-Harvesting I Complex
Authors: Young C. S. , Reyes R. C. , Beatty J. T.
Published: December 2019

Rhodobacter capsulatus ORF1696
mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the
ORF1696
gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of
ORF1696
resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when
trans
complemented with a plasmid expressing the
ORF1696
gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in
ORF1696
. We suggest that a
rho
-dependent transcription termination site exists between the
Mun
I and proximal
Stu
I sites of
ORF1696
. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the
ORF1696
parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.
Promoter Characterization and Constitutive Expression of the
Escherichia coli gcvR
Gene
Authors: Ghrist Angela C. , Stauffer George V.
Published: December 2019

The
Escherichia coli
glycine cleavage repressor protein (GcvR) negatively regulates expression of the glycine cleavage operon (
gcv
). In this study, the
gcvR
translational start site was determined by N-terminal amino acid sequence analysis of a GcvR-LacZ fusion protein. Primer extension analysis of the
gcvR
promoter region identified a primary transcription start site 27 bp upstream of the UUG translation start site and a minor transcription start site approximately 100 bp upstream of the translation start codon. The -10 and -35 promoter regions upstream of the primary transcription start site were defined by mutational analysis. Expression of a
gcvR-lacZ
fusion was unaltered in the presence of glycine or inosine, molecules known to induce or repress expression of
gcv
, respectively. In addition, it was shown that
gcvR-lacZ
expression is neither regulated by the glycine cleavage activator protein (GcvA) nor autogenously regulated by GcvR. From DNA sequence analysis, it was predicted that the translation start codon of the downstream
bcp
gene overlaps the
gcvR
stop codon, suggesting that these genes may form an operon. However, a down mutation in the -10 promoter region of
gcvR
had no effect on the expression of a downstream
bcp-lacZ
fusion, and primer extension analysis of the
bcp
promoter region demonstrated that
bcp
has its own promoter within the
gcvR
coding sequence. These results show that
gcvR
and
bcp
do not form an operon. Furthermore, the deletion of
bcp
from the chromosome had no effect on
gcv-lacZ
expression.
Acquisition of Five High-
M
r
Penicillin-Binding Protein Variants during Transfer of High-Level β-Lactam Resistance from
Streptococcus mitis
to
Streptococcus pneumoniae
Authors: Hakenbeck Regine , König Andrea , Kern Izabella , van der Linden Mark , Keck Wolfgang , Billot-Klein Danielle , Legrand Raymond , Schoot Bernard , Gutmann Laurent
Published: December 2019

Penicillin-resistant isolates of
Streptococcus pneumoniae
generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in β-lactam-resistant transformants of
S. pneumoniae
obtained with chromosomal donor DNA from a
Streptococcus mitis
isolate. Different lineages of β-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzylpenicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 μg/ml, respectively, close to the MICs for the
S. mitis
donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental
S. pneumoniae
strain. The results stress the potential for the acquisition by
S. pneumoniae
of high-level β-lactam resistance by interspecies gene transfer.
Chemotactic Adaptation Is Altered by Changes in the Carboxy-Terminal Sequence Conserved among the Major Methyl-Accepting Chemoreceptors
Authors: Okumura Hisashi , Nishiyama So-ichiro , Sasaki Akie , Homma Michio , Kawagishi Ikuro
Published: December 2019

In
Escherichia coli
and
Salmonella typhimurium
, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless
E. coli
strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.
ntn
Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in
Pseudomonas
sp. Strain TW3
Authors: James Keith D. , Williams Peter A.
Published: December 2019

Pseudomonas
sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes,
ntnWCMAB
*, which are very similar to and in the same order as the
xyl
operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (
ntnW
), an NAD
+
-linked benzaldehyde dehydrogenase (
ntnC
), a two-gene toluene monooxygenase (
ntnMA
), and part of a benzyl alcohol dehydrogenase (
ntnB
*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding
xylWCMAB
genes. The
xylB
homolog on the TW3 genome (
ntnB
*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway.
Variation of the Ribosomal Operon 16S-23S Gene Spacer Region in Representatives of
Salmonella enterica
Subspecies
Authors: Luz Sara Pérez , Rodríguez-Valera Francisco , Lan Ruiting , Reeves Peter R.
Published: December 2019

The 16S-23S spacer regions of two ribosomal operons (
rrnA
and
rrnE
) have been sequenced in seven representatives of the
Salmonella enterica
subspecies. Isolated nucleotide substitutions were found at the same sites as in
Escherichia coli
but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in
E. coli
, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of
rrnE
put subspecies I closer to
E. coli
K-12 than to the other
S. enterica
subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in
E. coli
, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family
Enterobacteriaceae
were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.
An AT-Rich Tract Containing an Integration Host Factor-Binding Domain and Two UP-Like Elements Enhances Transcription from the
pilEp
1
Promoter of
Neisseria gonorrhoeae
Authors: Fyfe Janet A. M. , Davies John K.
Published: December 2019

The
pilE
gene of
Neisseria gonorrhoeae
is transcribed from a ς
70
promoter (
pilEp
1
) with an AT-rich tract extending 65 nucleotides upstream of the −35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription from
pilEp
1
in
N. gonorrhoeae
. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both
N. gonorrhoeae
and an
Escherichia coli
strain producing IHF. In addition, an UP-like element positioned between the −35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the −35 box.
A
Bacillus subtilis
Gene Induced by Cold Shock Encodes a Membrane Phospholipid Desaturase
Authors: Aguilar Pablo S. , Cronan John E. , de Mendoza Diego
Published: December 2019

Bacillus subtilis
grown at 37°C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37°C are transferred to 20°C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of
B. subtilis
. This gene, called
des
, was isolated by complementation of
Escherichia coli
strains with mutations in either of two different genes of UFA synthesis. The
des
gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the
des
gene in
E. coli
resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA
cis
-5-hexadecenoic acid, indicating that the
B. subtilis des
gene product is a Δ5 acyl-lipid desaturase. The
des
gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The
des
null mutant strain grew as well as its congenic parent at 20 or 37°C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the
des
promoter directed the synthesis of a
lacZ
reporter gene showed that
des
expression is repressed at 37°C, but a shift of cultures from 37 to 20°C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.
Oxygen Regulation of the
ccoN
Gene Encoding a Component of the
cbb
3
Oxidase in
Rhodobacter sphaeroides
2.4.1
T
: Involvement of the FnrL Protein
Authors: Mouncey Nigel J. , Kaplan Samuel
Published: December 2019

The
ccoNOQP
gene cluster of
Rhodobacter sphaeroides
2.4.1
T
encodes a
cbb
3
cytochrome oxidase which is utilized in oxygen-limited conditions for aerobic respiration. The β-galactosidase activity of a
ccoN
::
lacZ
transcriptional fusion was low under high (30%)-oxygen and anaerobic growth conditions. Maximal
ccoN
::
lacZ
expression was observed when the oxygen concentration was lowered to 2%. In an FnrL mutant,
ccoN
::
lacZ
expression was significantly lower than in the wild-type strain, suggesting that FnrL is a positive regulator of genes encoding the
cbb
3
oxidase.
Regulation of Lactose Utilization Genes in
Staphylococcus xylosus
Authors: Bassias Joannis , Brückner Reinhold
Published: December 2019

The lactose utilization genes of
Staphylococcus xylosus
have been isolated and characterized. The system is comprised of two structural genes,
lacP
and
lacH
, encoding the lactose permease and the β-galactosidase proteins, respectively, and a regulatory gene,
lacR
, coding for an activator of the AraC/XylS family. The lactose utilization genes are divergently arranged, the
lacPH
genes being opposite to
lacR
. The
lacPH
genes are cotranscribed from one promoter in front of
lacP
, whereas
lacR
is transcribed from two promoters of different strengths. Lactose transport as well as β-galactosidase activity are inducible by the addition of lactose to the growth medium. Primer extension experiments demonstrated that regulation is achieved at the level of
lacPH
transcription initiation. Inducibility and efficient
lacPH
transcription are dependent on a functional
lacR
gene. Inactivation of
lacR
resulted in low and constitutive
lacPH
expression. Expression of
lacR
itself is practically constitutive, since transcription initiated at the major
lacR
promoter does not respond to the availability of lactose. Only the minor
lacR
promoter is lactose inducible. Apart from lactose-specific, LacR-dependent control, the
lacPH
promoter is also subject to carbon catabolite repression mediated by the catabolite control protein CcpA. When glucose is present in the growth medium,
lacPH
transcription initiation is reduced. Upon
ccpA
inactivation, repression at the
lacPH
promoter is relieved. Despite this loss of transcriptional regulation in the
ccpA
mutant strain, β-galactosidase activity is still reduced by glucose, suggesting another level of control.
Metalloadsorption by
Escherichia coli
Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB
Authors: Sousa Carolina , Kotrba Pavel , Ruml Tomas , Cebolla Angel , De Lorenzo Víctor
Published: December 2019

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in
Escherichia coli
as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of
Saccharomyces cerevisiae
(yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB
153
-YMT and LamB
153
-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of
E. coli
cells to bind Cd
2+
15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.
Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism inLactobacillus pentosus
Authors: Chaillou Stéphane , Lokman B. Christien , Leer Rob J. , Posthuma Clara , Postma Pieter W. , Pouwels Peter H.
Published: December 2019

Two genes,xylPandxylQ, from the xylose regulon ofLactobacillus pentosuswere cloned and sequenced. Together with the repressor gene of the regulon,xylR, thexylPQgenes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream ofxylP. A putativexylRbinding site (xylO) and acre-like element, mediating CcpA-dependent catabolite repression, were found in the promoter region.L. pentosusmutants in which bothxylPandxylQ(LPE1) or onlyxylQ(LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (α-d-xylopyranosyl-(1,6)-d-glucopyranose). Disruption ofxylQresulted specifically in the loss of a membrane-associated α-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria, α-xylosidase could catalyze the hydrolysis of isoprimeverose andp-nitrophenyl-α-d-xylopyranoside with apparentKmandVmaxvalues of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the α-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-α-d-xylopyranoside nor α-glucosides were substrates. Glucose repressed the synthesis of α-xylosidase fivefold, and 80% of this repression was released in anL. pentosus ΔccpAmutant. The α-xylosidase gene was also expressed in the absence of xylose whenxylRwas disrupted.
Structure and Expression of the FlaA Periplasmic Flagellar Protein ofBorrelia burgdorferi
Authors: Ge Yigong , Li Chunhao , Corum Linda , Slaughter Clive A. , Charon Nyles W.
Published: December 2019

The spirochete which causes Lyme disease,Borrelia burgdorferi, has many features common to other spirochete species. Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella (PFs). The PFs are subterminally attached to the cell cylinder and overlap in the center of the cell. Most descriptions of theB. burgdorferiflagellar filaments indicate that these organelles consist of only one flagellin protein (FlaB). In contrast, the PFs from other spirochete species are comprised of an outer layer of FlaA and a core of FlaB. We recently found that aflaAhomolog was expressed inB. burgdorferiand that it mapped in afla/cheoperon. These results led us to analyze the PFs and FlaA ofB. burgdorferiin detail. Using Triton X-100 to remove the outer membrane and isolate the PFs, we found that the 38.0-kDa FlaA protein purified with the PFs in association with the 41.0-kDa FlaB protein. On the other hand, purifying the PFs by using Sarkosyl resulted in no FlaA in the isolated PFs. Sarkosyl has been used by others to purifyB. burgdorferiPFs, and our results explain in part their failure to find FlaA. Unlike other spirochetes,B. burgdorferiFlaA was expressed at a lower level than FlaB. In characterizing FlaA, we found that it was posttranslationally modified by glycosylation, and thus it resembles its counterpart fromSerpulina hyodysenteriae. We also tested if FlaA was synthesized in a spontaneously occurring PF mutant ofB. burgdorferi(HB19Fla−). Although this mutant still synthesizedflaAmessage in amounts similar to the wild-type amounts, it failed to synthesize FlaA protein. These results suggest that, in agreement with data found for FlaB and other spirochete flagellar proteins, FlaA is likely to be regulated on the translational level. Western blot analysis usingTreponema pallidumanti-FlaA serum indicated that FlaA was antigenically well conserved in several spirochete species. Taken together, the results indicate that both FlaA and FlaB comprise the PFs ofB. burgdorferiand that they are regulated differently from flagellin proteins of other bacteria.
Isolation of an
Escherichia coli
K-12 Mutant Strain Able To Form Biofilms on Inert Surfaces: Involvement of a New
ompR
Allele That Increases Curli Expression
Authors: Vidal Olivier , Longin Robert , Prigent-Combaret Claire , Dorel Corinne , Hooreman Michel , Lejeune Philippe
Published: December 2019

Classical laboratory strains of
Escherichia coli
do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized
E. coli
K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652–655, 1989). The production of curli (visualized by Congo red binding) and the expression of the
csgA
gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the
ompR
allele described in this work. Transduction of knockout mutations in either
csgA
or
ompR
caused the loss of the adherence properties of several biofilm-forming
E. coli
strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.
Regulation of Benzoate Degradation in
Acinetobacter
sp. Strain ADP1 by BenM, a LysR-Type Transcriptional Activator
Authors: Collier Lauren S. , Gaines George L. , Neidle Ellen L.
Published: December 2019

In
Acinetobacter
sp. strain ADP1, benzoate degradation requires the
ben
genes for converting benzoate to catechol and the
cat
genes for degrading catechol. Here we describe a novel transcriptional activator, BenM, that regulates the chromosomal
ben
and
cat
genes. BenM is homologous to CatM, a LysR-type transcriptional activator of the
cat
genes. Unusual regulatory features of this system include the abilities of both BenM and CatM to recognize the same inducer,
cis
,
cis
-muconate, and to regulate some of the same genes, such as
catA
and
catB
. Unlike CatM, BenM responded to benzoate. Benzoate together with
cis
,
cis
-muconate increased the BenM-dependent expression of the
benABCDE
operon synergistically. CatM was not required for this synergism, nor did CatM regulate the expression of a chromosomal
benA
::
lacZ
transcriptional fusion. BenM-mediated regulation differs significantly from that of the TOL plasmid-encoded conversion of benzoate to catechol in pseudomonads. The
benM
gene is immediately upstream of, and divergently transcribed from,
benA
, and a possible DNA binding site for BenM was identified between the two coding regions. Two mutations in the predicted operator/promoter region rendered
ben
gene expression either constitutive or inducible by
cis
,
cis
-muconate but not benzoate. Mutants lacking BenM, CatM, or both of these regulators degraded aromatic compounds at different rates, and the levels of intermediary metabolites that accumulated depended on the genetic background. These studies indicated that BenM is necessary for
ben
gene expression but not for expression of the
cat
genes, which can be regulated by CatM. In a
catM
-disrupted strain, BenM was able to induce higher levels of
catA
expression than
catB
expression.
A Seven-Gene Locus for Synthesis of Phenazine-1-Carboxylic Acid by
Pseudomonas fluorescens
2-79
Authors: Mavrodi Dmitri V. , Ksenzenko Vladimir N. , Bonsall Robert F. , Cook R. James , Boronin Alexander M. , Thomashow Linda S.
Published: December 2019

Pseudomonas fluorescens
2-79 produces the broad-spectrum antibiotic phenazine-1-carboxylic acid (PCA), which is active against a variety of fungal root pathogens. In this study, seven genes designated
phzABCDEFG
that are sufficient for synthesis of PCA were localized within a 6.8-kb
Bgl
II-
Xba
I fragment from the phenazine biosynthesis locus of strain 2-79. Polypeptides corresponding to all
phz
genes were identified by analysis of recombinant plasmids in a T7 promoter/polymerase expression system. Products of the
phzC
,
phzD
, and
phzE
genes have similarities to enzymes of shikimic acid and chorismic acid metabolism and, together with PhzF, are absolutely necessary for PCA production. PhzG is similar to pyridoxamine-5′-phosphate oxidases and probably is a source of cofactor for the PCA-synthesizing enzyme(s). Products of the
phzA
and
phzB
genes are highly homologous to each other and may be involved in stabilization of a putative PCA-synthesizing multienzyme complex. Two new genes,
phzX
and
phzY
, that are homologous to
phzA
and
phzB
, respectively, were cloned and sequenced from
P. aureofaciens
30-84, which produces PCA, 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxyphenazine. Based on functional analysis of the
phz
genes from strains 2-79 and 30-84, we postulate that different species of fluorescent pseudomonads have similar genetic systems that confer the ability to synthesize PCA.
3-Nitroadipate, a Metabolic Intermediate for Mineralization of 2,4-Dinitrophenol by a New Strain of a
Rhodococcus
Species
Authors: Blasco Rafael , Moore Edward , Wray Victor , Pieper Dietmar , Timmis Kenneth , Castillo Francisco
Published: December 2019

The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a
Rhodococcus
species closely related to
Rhodococcus opacus. Rhodococcus
sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by
1
H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.
Function and Regulation of
glnA
in the Methanogenic Archaeon
Methanococcus maripaludis
Authors: Cohen-Kupiec Rachel , Marx Christopher J. , Leigh John A.
Published: December 2019

The
glnA
gene in the domains
Bacteria
and
Archaea
encodes glutamine synthetase, a universally distributed enzyme that functions in ammonia assimilation and glutamine synthesis. We investigated the regulation and function of
glnA
in the methanogenic archaeon
Methanococcus maripaludis
. The deduced amino acid sequence of the gene demonstrated its membership in class GSI-α of glutamine synthetases. The gene appeared to be expressed as a monocistronic operon.
glnA
mRNA levels and specific activities of glutamine synthetase were regulated similarly by nitrogen. Three transcription start sites were identified, corresponding to two overlapping nitrogen-regulated promoters and one weaker constitutive promoter. An inverted repeat immediately upstream of the regulated transcription start sites mediated repression under noninducing conditions. Thus, mutations that altered the sequence of the inverted repeat resulted in derepression. The inverted repeat had sequence similarity with a repeat that we previously identified as the
nif
operator of
M. maripaludis
, suggesting a common mechanism of nitrogen regulation. Efforts to produce a
glnA
null mutant failed, suggesting that
glnA
is an essential gene in
M. maripaludis
.
Peptidoglycan Fine Structure of the Radiotolerant Bacterium
Deinococcus radiodurans
Sark
Authors: Quintela José Carlos , García-del Portillo Francisco , Pittenauer Ernst , Allmaier Günter , de Pedro Miguel A.
Published: December 2019

Peptidoglycan from
Deinococcus radiodurans
was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was:
N
-acetylglucosamine–
N
-acetylmuramic acid–
l
-Ala–
d
-Glu-(γ)–
l
-Orn-[(δ)Gly-Gly]–
d
-Ala–
d
-Ala. Cross-linkage was mediated by (Gly)
2
bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close
Thermus thermophilus
are discussed.
Roles of Chemosensory Pathways in Transient Changes in Swimming Speed of
Rhodobacter sphaeroides
Induced by Changes in Photosynthetic Electron Transport
Authors: Romagnoli Simona , Armitage Judith P.
Published: December 2019

The response of free-swimming
Rhodobacter sphaeroides
to increases and decreases in the intensity of light of different wavelengths was analyzed. There was a transient (1 to 2 s) increase in swimming speed in response to an increase in light intensity, and there was a similar transient stop when the light intensity decreased. Measurement of changes in membrane potential and the use of electron transport inhibitors showed that the transient increase in swimming speed, following an increase in light intensity, and the stop following its decrease were the result of changes in photosynthetic electron transport.
R. sphaeroides
has two operons coding for multiple homologs of the enteric chemosensory genes. Mutants in the first chemosensory operon showed wild-type photoresponses. Mutants with the
cheA
gene of the second operon (
cheA
II
) deleted, either with or without the first operon present, showed inverted photoresponses, with free-swimming cells stopping on an increase in light intensity and increasing swimming speed on a decrease. These mutants also lacked adaptation. Transposon mutants with mutations in
cheA
II
, which also reduced expression of downstream genes, however, showed no photoresponses. These results show that (i) free-swimming cells respond to both an increase and a decrease in light intensity (tethered cells only show the stopping on a step down in light intensity), (ii) the signal comes from photosynthetic electron transfer, and (iii) the signal is primarily channelled through the second chemosensory pathway. The different responses shown by the
cheA
II
deletion and insertion mutants suggest that CheW
II
is required for photoresponses, and a third sensory pathway can substitute for CheA
II
as long as CheW
II
is present. The inverted response suggests that transducers are involved in photoresponses as well as chemotactic responses.
Cloning and Expression of Three New
Azotobacter vinelandii
Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases
Authors: Svanem Britt Iren Glærum , Skjåk-Bræk Gudmund , Ertesvåg Helga , Valla Svein
Published: December 2019

The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from
Azotobacter vinelandii
(
algE1
to -
5
) has previously been reported. The corresponding proteins catalyze the Ca
2+
-dependent polymer-level epimerization of β-
d
-mannuronic acid to α-
l
-guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated
algE6
,
algE7
, and
algY
. All three genes were sequenced and expressed in
Escherichia coli
. AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in
algE4
expressed an active epimerase. Southern blot analysis of genomic
A. vinelandii
DNA, using the 5′ part of
algE2
as a probe, indicated that all hybridization signals originated from
algE1
to -
5
or the three new genes reported here.
Phosphate Control of Oxytetracycline Production by
Streptomyces rimosus
Is at the Level of Transcription from Promoters Overlapped by Tandem Repeats Similar to Those of the DNA-Binding Sites of the OmpR Family
Authors: McDowall Kenneth J. , Thamchaipenet Arinthip , Hunter Iain S.
Published: December 2019

Physiological studies have shown that
Streptomyces rimosus
produces the polyketide antibiotic oxytetracycline abundantly when its mycelial growth is limited by phosphate starvation. We show here that transcripts originating from the promoter for one of the biosynthetic genes,
otcC
(encoding anhydrotetracycline oxygenase), and from a promoter for the divergent
otcX
genes peak in abundance at the onset of antibiotic production induced by phosphate starvation, indicating that the synthesis of oxytetracycline is controlled, at least in part, at the level of transcription. Furthermore, analysis of the sequences of the promoters for
otcC
,
otcX
, and the polyketide synthase (
otcY
) genes revealed tandem repeats having significant similarity to the DNA-binding sites of ActII-Orf4 and DnrI, which are
Streptomyces
antibiotic regulatory proteins (SARPs) related to the OmpR family of transcription activators. Together, the above results suggest that oxytetracycline production by
S. rimosus
requires a SARP-like transcription factor that is either produced or activated or both under conditions of low phosphate concentrations. We also provide evidence consistent with the
otrA
resistance gene being cotranscribed with
otcC
as part of a polycistronic message, suggesting a simple mechanism of coordinate regulation which ensures that resistance to the antibiotic increases in proportion to production.
MHY1
Encodes a C
2
H
2
-Type Zinc Finger Protein That Promotes Dimorphic Transition in the Yeast
Yarrowia lipolytica
Authors: Hurtado Cleofe A. R. , Rachubinski Richard A.
Published: December 2019

The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi. Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions. We have isolated and characterized a gene,
MHY1
, whose transcription is dramatically increased during the yeast-to-hypha transition in
Yarrowia lipolytica
. Deletion of
MHY1
is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth.
MHY1
encodes a C
2
H
2
-type zinc finger protein, Mhy1p, which can bind putative
cis
-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor. Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip.
Escherichia coli
DNA Topoisomerase I Copurifies with Tn
5
Transposase, and Tn
5
Transposase Inhibits Topoisomerase I
Authors: Yigit Hesna , Reznikoff William S.
Published: December 2019

Tn
5
transposase (Tnp) overproduction is lethal to
Escherichia coli
. Genetic evidence suggested that this killing involves titration of
E. coli
topoisomerase I (Topo I). Here, we present biochemical evidence that supports this model. Tn
5
Tnp copurifies with Topo I while nonkilling derivatives of Tnp, Δ37Tnp and Δ55Tnp (Inhibitor [Inh]), show reduced affinity or no affinity, respectively, for Topo I. In agreement with these results, the presence of Tnp, but not Δ37 or Inh derivatives of Tnp, inhibits the DNA relaxation activity of Topo I in vivo as well as in vitro. Other proteins, including RNA polymerase, are also found to copurify with Tnp. For RNA polymerase, reduced copurification with Tnp is observed in extracts from a
topA
mutant strain, suggesting that RNA polymerase interacts with Topo I and not Tnp.