bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron-Responsive Gene Regulation in a
Campylobacter jejuni fur
Mutant
Authors: van Vliet Arnoud H. M. , Wooldridge Karl G. , Ketley Julian M.
Published: December 2019

The expression of iron-regulated systems in gram-negative bacteria is generally controlled by the Fur protein, which represses the transcription of iron-regulated promoters by using Fe
2+
as a cofactor. Mutational analysis of the
Campylobacter jejuni fur
gene was carried out by generation of a set of mutant copies of
fur
which had a kanamycin or chloramphenicol resistance gene introduced into the regions encoding the N and C termini of the Fur protein. The mutated genes were recombined into the
C. jejuni
NCTC 11168 chromosome, and putative mutants were confirmed by Southern hybridization.
C. jejuni
mutants were obtained only when the resistance genes were transcribed in the same orientation as the
fur
gene. The
C. jejuni fur
mutant grew slower than the parental strain. Comparison of protein profiles of fractionated
C. jejuni
cells grown in low- or high-iron medium indicated derepressed expression of three iron-regulated outer membrane proteins with molecular masses of 70, 75, and 80 kDa. Characterization by N-terminal amino acid sequencing showed the 75-kDa protein to be identical to CfrA, a
Campylobacter coli
siderophore receptor homologue, whereas the 70-kDa protein was identified as a new siderophore receptor homologue. Periplasmic fractions contained four derepressed proteins with molecular masses of 19, 29, 32, and 36 kDa. The 19-kDa protein has been previously identified, but its function is unknown. The cytoplasmic fraction contained two iron-repressed and two iron-induced proteins with molecular masses of 26, 55, 31, and 40 kDa, respectively. The two iron-repressed proteins have been previously identified as the oxidative stress defense proteins catalase (KatA) and alkyl hydroperoxide reductase (AhpC). AhpC and KatA were still iron regulated in the
fur
mutant, suggesting the presence of Fur-independent iron regulation. Further analysis of the
C. jejuni
iron and Fur regulons by using two-dimensional gel electrophoresis demonstrated the total number of iron- and Fur-regulated proteins to be lower than for other bacterial pathogens.
Identification of
Candida albicans ALS2
and
ALS4
and Localization of Als Proteins to the Fungal Cell Surface
Authors: Hoyer L. L. , Payne T. L. , Hecht J. E.
Published: December 2019

Additional genes in the growing
ALS
family of
Candida albicans
were isolated by PCR screening of a genomic fosmid library with primers designed from the consensus tandem-repeat sequence of
ALS1
. This procedure yielded fosmids encoding
ALS2
and
ALS4. ALS2
and
ALS4
conformed to the three-domain structure of
ALS
genes, which consists of a central domain of tandemly repeated copies of a 108-bp motif, an upstream domain of highly conserved sequences, and a domain of divergent sequences 3′ of the tandem repeats. Alignment of five predicted Als protein sequences indicated conservation of N- and C-terminal hydrophobic regions which have the hallmarks of secretory signal sequences and glycosylphosphatidylinositol addition sites, respectively. Heterologous expression of an N-terminal fragment of Als1p in
Saccharomyces cerevisiae
demonstrated function of the putative signal sequence with cleavage following Ala17. This signal sequence cleavage site was conserved in the four other Als proteins analyzed, suggesting identical processing of each protein. Primary-structure features of the five Als proteins suggested a cell-surface localization, which was confirmed by indirect immunofluorescence with an anti-Als antiserum. Staining was observed on mother yeasts and germ tubes, although the intensity of staining on the mother yeast decreased with elongation of the germ tube. Similar to other
ALS
genes,
ALS2
and
ALS4
were differentially regulated.
ALS4
expression was correlated with the growth phase of the culture;
ALS2
expression was not observed under many different in vitro growth conditions. The data presented here demonstrate that
ALS
genes encode cell-surface proteins and support the conclusion that the size and number of Als proteins on the
C. albicans
cell surface vary with strain and growth conditions.
Novel
rkp
Gene Clusters of
Sinorhizobium meliloti
Involved in Capsular Polysaccharide Production and Invasion of the Symbiotic Nodule: the
rkpK
Gene Encodes a UDP-Glucose Dehydrogenase
Authors: Kereszt Attila , Kiss Ernő , Reuhs Bradley L. , Carlson Russell W. , Kondorosi Ádám , Putnoky Péter
Published: December 2019

The production of exopolysaccharide (EPS) was shown to be required for the infection process by rhizobia that induce the formation of indeterminate nodules on the roots of leguminous host plants. In
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
) Rm41, a capsular polysaccharide (KPS) analogous to the group II K antigens of
Escherichia coli
can replace EPS during symbiotic nodule development and serve as an attachment site for the strain-specific bacteriophage φ16-3. The
rkpA
to -
J
genes in the chromosomal
rkp-1
region code for proteins that are involved in the synthesis, modification, and transfer of an as-yet-unknown lipophilic molecule which might function as a specific lipid carrier during KPS biosynthesis. Here we report that with a phage φ16-3-resistant population obtained after random Tn
5
mutagenesis, we have identified novel mutants impaired in KPS production by genetic complementation and biochemical studies. The mutations represent two novel loci, designated the
rkp-2
and
rkp-3
regions, which are required for the synthesis of rhizobial KPS. The
rkp-2
region harbors two open reading frames (ORFs) organized in monocistronic transcription units. Although both genes are required for normal lipopolysaccharide production, only the second one, designated
rkpK
, is involved in the synthesis of KPS. We have demonstrated that RkpK possesses UDP-glucose dehydrogenase activity, while the protein product of ORF1 might function as a UDP-glucuronic acid epimerase.
The 20S Proteasome of
Streptomyces coelicolor
Authors: Nagy István , Tamura Tomohiro , Vanderleyden Jos , Baumeister Wolfgang , De Mot René
Published: December 2019

20S proteasomes were purified from
Streptomyces coelicolor
A3(2) and shown to be built from one α-type subunit (PrcA) and one β-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the
Streptomyces
metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (
prcBA
) similar to
Rhodococcus
and
Mycobacterium
spp. and showed that the β subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved
orf7
and an AAA ATPase gene (
arc
).
Efficiency of the pTF-FC2
pas
Poison-Antidote Stability System in
Escherichia coli
Is Affected by the Host Strain, and Antidote Degradation Requires the Lon Protease
Authors: Smith Anthony S. G. , Rawlings Douglas E.
Published: December 2019

The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (
pas
) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in
Escherichia coli
CSH50, a 2.5-fold increase in
E. coli
JM105, and no detectable stabilization in
E. coli
strains JM107 and JM109. The lethality of the PasB toxin was far higher in the
E. coli
strains in which the
pas
was most effective. Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin. A decrease in host cell death following plasmid loss from an
E. coli lon
mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.
Functional Consequences of Changing Proline Residues in the Phenylalanine-Specific Permease of
Escherichia coli
Authors: Pi Jing , Dogovski C. , Pittard A. J.
Published: December 2019

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium
Escherichia coli
. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650–2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [
35
S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.
Molecular Genetic Analysis of Phosphite and Hypophosphite Oxidation by
Pseudomonas stutzeri
WM88
Authors: Metcalf William W. , Wolfe Ralph S.
Published: December 2019

The first molecular and genetic characterization of a biochemical pathway for oxidation of the reduced phosphorus (P) compounds phosphite and hypophosphite is reported. The pathway was identified in
Pseudomonas stutzeri
WM88, which was chosen for detailed studies from a group of organisms isolated based on their ability to oxidize hypophosphite (+1 valence) and phosphite (+3 valence) to phosphate (+5 valence). The genes required for oxidation of both compounds by
P. stutzeri
WM88 were cloned on a single ca. 30-kbp DNA fragment by screening for expression in
Escherichia coli
and
Pseudomonas aeruginosa
. Two lines of evidence suggest that hypophosphite is oxidized to phosphate via a phosphite intermediate. First, plasmid subclones that conferred oxidation of phosphite, but not hypophosphite, upon heterologous hosts were readily obtained. All plasmid subclones that failed to confer phosphite oxidation also failed to confer hypophosphite oxidation. No subclones that conferred only hypophosphite expression were obtained. Second, various deletion derivatives of the cloned genes were made in vitro and recombined onto the chromosome of
P. stutzeri
WM88. Two phenotypes were displayed by individual mutants. Mutants with the region encoding phosphite oxidation deleted (based upon the subcloning results) lost the ability to oxidize either phosphite or hypophosphite. Mutants with the region encoding hypophosphite oxidation deleted lost only the ability to oxidize hypophosphite. The phenotypes displayed by these mutants also demonstrate that the cloned genes are responsible for the P oxidation phenotypes displayed by the original
P. stutzeri
WM88 isolate. The DNA sequences of the minimal regions implicated in oxidation of each compound were determined. The region required for oxidation of phosphite to phosphate putatively encodes a binding-protein-dependent phosphite transporter, an NAD
+
-dependent phosphite dehydrogenase, and a transcriptional activator of the
lysR
family. The region required for oxidation of hypophosphite to phosphite putatively encodes a binding-protein-dependent hypophosphite transporter and an α-ketoglutarate-dependent hypophosphite dioxygenase. The finding of genes dedicated to oxidation of reduced P compounds provides further evidence that a redox cycle for P may be important in the metabolism of this essential, and often growth-limiting, nutrient.
Domain Analysis of the FliM Protein of
Escherichia coli
Authors: Mathews Michael A. A. , Tang Hua Lucy , Blair David F.
Published: December 2019

The FliM protein of
Escherichia coli
is required for the assembly and function of flagella. Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including FliN, FliG, phosphorylated CheY, other copies of FliM, and possibly MotA and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C-terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the rest of FliM by a segment (residues 237 to 247) that is poorly conserved, tolerates linker insertion, and may be an interdomain linker. Binding to FliG occurs through multiple segments of FliM, some in the C-terminal domain and others in an N-terminal domain of 144 residues. Binding of FliM to CheY and phospho-CheY was complex. In coprecipitation experiments using purified FliM, the protein bound weakly to unphosphorylated CheY and more strongly to phospho-CheY, in agreement with previous reports. By contrast, in experiments using FliM in fresh cell lysates, the protein bound to unphosphorylated CheY about as well as to phospho-CheY. Determinants for binding CheY occur both near the N terminus of FliM, which appears most important for binding to the phosphorylated protein, and in the C-terminal domain, which binds more strongly to unphosphorylated CheY. Several different deletions and linker insertions in FliM enhanced its binding to phospho-CheY in coprecipitation experiments with protein from cell lysates. This suggests that determinants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangement and function of FliM domains in the flagellar motor.
Gigantism in a Bacterium,Epulopiscium fishelsoni, Correlates with Complex Patterns in Arrangement, Quantity, and Segregation of DNA
Authors: Bresler V. , Montgomery W. L. , Fishelson L. , Pollak P. E.
Published: December 2019

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2,000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of ∼30 μm to >500 μm. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and “pinching” of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines.
The
Saccharomyces cerevisiae
GATA Factors Dal80p and Deh1p Can Form Homo- and Heterodimeric Complexes
Authors: Svetlov Vladimir V. , Cooper Terrance G.
Published: December 2019

GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in
Saccharomyces cerevisiae
. Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences. A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer. Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini. Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays. We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form. Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers. If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors.
Methylation of FrzCD Defines a Discrete Step in the Developmental Program of
Myxococcus xanthus
Authors: Geng Yongzhi , Yang Zhaomin , Downard John , Zusman David , Shi Wenyuan
Published: December 2019

Myxococcus xanthus
is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The
frz
signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized
asg
,
bsg
,
csg
, and
esg
mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of
M. xanthus
and that social motility mutants are not blocked in these early developmental steps.
DdlN from Vancomycin-Producing
Amycolatopsis orientalis
C329.2 Is a VanA Homologue with
d
-Alanyl-
d
-Lactate Ligase Activity
Authors: Marshall C. Gary , Wright Gerard D.
Published: December 2019

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in
d
-alanyl-
d
-lactate. A key enzyme in this process is a
d
-alanyl-
d
-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide
d
-alanyl-
d
-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism
Amycolatopsis orientalis
C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for
d
-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.
Molecular Cloning and Characterization of Tap, a Putative Multidrug Efflux Pump Present inMycobacterium fortuitumandMycobacterium tuberculosis
Authors: Aínsa José A. , Blokpoel Marian C. J. , Otal Isabel , Young Douglas B. , De Smet Koen A. L. , Martín Carlos
Published: December 2019

A recombinant plasmid isolated from aMycobacterium fortuitumgenomic library by selection for gentamicin and 2-N′-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced intoM. smegmatis. Further characterization of this plasmid allowed the identification of theM. fortuitum tapgene. A homologous gene in theM. tuberculosisH37Rv genome has been identified. TheM. tuberculosis tapgene (Rv1258 in the annotated sequence of theM. tuberculosisgenome) was cloned and conferred low-level resistance to tetracycline when introduced intoM. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanidem-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing thetapgenes were decreased. When tetracycline accumulation experiments were carried out with theM. fortuitum tapgene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organismM. fortuitumand the important pathogenM. tuberculosisare probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.
Characterization of a Peroxide-Resistant Mutant of the Anaerobic Bacterium
Bacteroides fragilis
Authors: Rocha Edson R. , Smith C. Jeffrey
Published: December 2019

A
Bacteroides fragilis
mutant resistant to hydrogen peroxide and alkyl peroxide was isolated by enrichment in increasing concentrations of hydrogen peroxide. The mutant strain was constitutively resistant to 100 mM H
2
O
2
and 5 mM cumene hydroperoxide (15-min exposure). In contrast, the parent strain was protected against <10 mM H
2
O
2
when the peroxide response was induced with a sublethal concentration of H
2
O
2
, and no protection was observed in untreated cells. In addition, catalase activity in the mutant strain was not repressed in anaerobic cultures as reported previously for the parent strain. Comparison of the protein profile of crude extracts of the
B. fragilis
strains revealed that at least three oxidative stress-induced proteins in the parent strain were constitutively expressed in the mutant as detected by nondenaturing polyacrylamide gel electrophoresis. N-terminal amino acid sequence of these overexpressed proteins confirmed the presence of a deregulated catalase (KatB), an alkyl hydroperoxidase reductase subunit C (AhpC), and a Dps/PexB homologue. Northern blot analysis and
katB
::
cat
transcriptional fusion studies revealed that in the mutant,
katB
was deregulated compared to the parent and that
katB
was controlled by a
trans
-acting regulatory mechanism. Moreover, constitutive expression of KatB and of the AhpC and Dps homologues in the H
2
O
2
-resistant mutant suggests that these proteins may share a common oxidative stress transcriptional regulator and may be involved in
B. fragilis
peroxide resistance.
Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences ingutandpapOperons
Authors: van der Woude Marjan , Hale W. Bradley , Low David A.
Published: December 2019

Most of the adenine residues in GATC sequences in theEscherichia colichromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) topapDNA was sufficient to protect the two GATC sequences in thepapregulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABDoperon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of thegutABDoperon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated thatgutABDgene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepapGATC-I and GATC-II sites.
Decay of
ermC
mRNA in a Polynucleotide Phosphorylase Mutant of
Bacillus subtilis
Authors: Bechhofer David H. , Wang Wei
Published: December 2019

ermC
mRNA decay was examined in a mutant of
Bacillus subtilis
that has a deleted
pnpA
gene (coding for polynucleotide phosphorylase). 5′-proximal RNA fragments less than 400 nucleotides in length were abundant in the
pnpA
strain but barely detectable in the wild type. On the other hand, the patterns of 3′-proximal RNA fragments were similar in the wild-type and
pnpA
strains. Northern blot analysis with different probes showed that the 5′ end of the decay intermediates was the native
ermC
5′ end. For one prominent
ermC
RNA fragment, in particular, it was shown that formation of its 3′ end was directly related to the presence of a stalled ribosome. 5′-proximal decay intermediates were also detected for transcripts encoded by the
yybF
gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.
Fitness Landscapes for Effects of Shape on Chemotaxis and Other Behaviors of Bacteria
Authors: Dusenbery David B.
Published: December 2019

Data on the shapes of 218 genera of free-floating or free-swimming bacteria reveal groupings around spherical shapes and around rod-like shapes of axial ratio about 3. Motile genera are less likely to be spherical and have larger axial ratios than nonmotile genera. The effects of shape on seven possible components of biological fitness were determined, and actual fitness landscapes in phenotype space are presented. Ellipsoidal shapes were used as models, since their hydrodynamic drag coefficients can be rigorously calculated in the world of low Reynolds number, where bacteria live. Comparing various shapes of the same volume, and assuming that departures from spherical have a cost that varies with the minimum radius of curvature, led to the following conclusions. Spherical shapes have the largest random dispersal by Brownian motion. Increased surface area occurs in oblate ellipsoids (disk-like), which rarely occur. Elongation into prolate ellipsoids (rod-like) reduces sinking speed, and this may explain why some nonmotile genera are rod-like. Elongation also favors swimming efficiency (to a limited extent) and the ability to detect stimulus gradients by any of three mechanisms. By far the largest effect (several hundred-fold) is on temporal detection of stimulus gradients, and this explains why rod-like shapes and this mechanism of chemotaxis are common.
Physical and Genetic Map of the
Pasteurella multocida
A:1 Chromosome
Authors: Hunt Meredith L. , Ruffolo Carmel G. , Rajakumar Kumar , Adler Ben
Published: December 2019

A physical and genetic map of the
Pasteurella multocida
A:1 genome was generated by using the restriction enzymes
Apa
I,
Ceu
I, and
Not
I. The positions of 23 restriction sites and 32 genes, including 5
rrn
operons, were localized on the 2.35-Mbp single circular chromosome. This report presents the first genetic and physical map for this genus.
mmr
, a
Mycobacterium tuberculosis
Gene Conferring Resistance to Small Cationic Dyes and Inhibitors
Authors: De Rossi Edda , Branzoni Manuela , Cantoni Rita , Milano Anna , Riccardi Giovanna , Ciferri Orio
Published: December 2019

The
mmr
gene, cloned from
Mycobacterium tuberculosis
, was shown to confer to
Mycobacterium smegmatis
resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [
3
H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP.
Mutational Analysis of theEscherichia coliK-12 TolA N-Terminal Region and Characterization of Its TolQ-Interacting Domain by Genetic Suppression
Authors: Germon Pierre , Clavel Thierry , Vianney Anne , Portalier Raymond , Lazzaroni Jean Claude
Published: December 2019

The Tol-Pal proteins ofEscherichia coliare involved in maintaining outer membrane integrity. They form two complexes in the cell envelope. Transmembrane domains of TolQ, TolR, and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. The N-terminal transmembrane domain of TolA anchors the protein to the cytoplasmic membrane and interacts with TolQ and TolR. Extensive mutagenesis of the N-terminal part of TolA was carried out to characterize the residues involved in such processes. Mutations affecting the function of TolA resulted in a lack or an alteration in TolA-TolQ or TolR-TolA interactions but did not affect the formation of TolQ-TolR complexes. Our results confirmed the importance of residues serine 18 and histidine 22, which are part of an SHLS motif highly conserved in the TolA and the related TonB proteins from different organisms. Genetic suppression experiments were performed to restore the functional activity of sometolAmutants. The suppressor mutations all affected the first transmembrane helix of TolQ. These results confirmed the essential role of the transmembrane domain of TolA in triggering interactions with TolQ and TolR.
The
arcABDC
Gene Cluster, Encoding the Arginine Deiminase Pathway of
Bacillus licheniformis
, and Its Activation by the Arginine Repressor ArgR
Authors: Maghnouj Abdelouahid , de Sousa Cabral Tiago Franco , Stalon Victor , Vander Wauven Corinne
Published: December 2019

The arginine deiminase pathway enables
Bacillus licheniformis
to grow anaerobically on arginine. Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway. In this study we have cloned and sequenced the
arc
genes encoding the pathway. They appear clustered in an operon-like structure in the order
arcA
(arginine deiminase),
arcB
(ornithine carbamoyltransferase),
arcD
(putative arginine-ornithine antiporter),
arcC
(carbamate kinase). It was found that
B. licheniformis
has an arginine repressor, ArgR, homologous to the
B. subtilis
arginine repressor AhrC. Mutants affected in
argR
were isolated. These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway. Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the
arc
promoter. The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.
Suppression of an Hsp70 Mutant Phenotype in
Saccharomyces cerevisiae
through Loss of Function of the Chromatin Component Sin1p/Spt2p
Authors: Baxter Bonnie K. , Craig Elizabeth A.
Published: December 2019

The Ssa subfamily of Hsp70 molecular chaperones in the budding yeast
Saccharomyces cerevisiae
has four members, encoded by
SSA1
,
SSA2
,
SSA3
, and
SSA4
. Deletion of the two constitutively expressed genes,
SSA1
and
SSA2
, results in cells which are slow growing and temperature sensitive. In this study, we demonstrate that an extragenic suppressor of the temperature sensitivity of
ssa1 ssa2
strains,
EXA1-1
, is a loss-of-function mutation in
SIN1/SPT2
, which encodes a nonhistone component of chromatin. Loss of function of Sin1p leads to overexpression of
SSA3
in the
ssa1 ssa2
mutant background, at a level which is sufficient to mediate suppression. In a strain which is wild type for
SSA
genes, we detected no effect of Sin1p on Ssa3p expression except under conditions of heat shock. Existing data indicate that expression of
SSA3
in the
ssa1 ssa2
mutant background as well as in heat-shocked wild-type strains is mediated by the heat shock transcription factor HSF. Our findings suggest that it is HSF-mediated induction of
SSA3
which is modulated by Sin1p. The
EXA1-1
suppressor mutation thus improves the growth of
ssa1 ssa2
strains by selectively increasing HSF-mediated expression of
SSA3
.
Analysis of Outgrowth of
Bacillus subtilis
Spores Lacking Penicillin-Binding Protein 2a
Authors: Murray Thomas , Popham David L. , Pearson Christine B. , Hand Arthur R. , Setlow Peter
Published: December 2019

The loss of
Bacillus subtilis
penicillin-binding protein (PBP) 2a, encoded by
pbpA
, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in
pbpA
spore outgrowth have shown that (i) outgrowing
pbpA
spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of
pbpA
spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and
pbpA
spores once cell elongation had been initiated; (iii) outgrowth and elongation of
pbpA
spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.
Studies of Regulation of Expression of the Propionate (
prpBCDE
) Operon Provide Insights into How
Salmonella typhimurium
LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways
Authors: Tsang Allen W. , Horswill Alexander R. , Escalante-Semerena Jorge C.
Published: December 2019

Expression of the
prpBCDE
operon of
Salmonella typhimurium
LT2 required (i) the synthesis of propionyl-coenzyme A (CoA) by the PrpE protein or the acetyl-CoA-synthesizing systems of the cell and (ii) the synthesis of 2-methylcitrate from propionyl-CoA and oxaloacetate by the PrpC protein. We propose that either 2-methylcitrate or a derivative of it signals the presence of propionate in the environment. This as yet unidentified signal is thought to serve as a coregulator of the activity of PrpR, the member of the sigma-54 family of transcriptional activators needed for activation of
prpBCDE
transcription. The CobB protein was also required for expression of the
prpBCDE
operon, but its role is less well understood. Expression of the
prpBCDE
operon in
cobB
mutants was restored to wild-type levels upon induction of the propanediol utilization (
pdu
) operon by 1,2-propanediol. This effect did not require catabolism of 1,2-propanediol, suggesting that a Pdu protein, not a catabolite of 1,2-propanediol, was responsible for the observed effect. We explain the existence of these redundant functions in terms of metabolic pathway integration. In an environment with 1,2-propanediol as the sole carbon and energy source, expression of the
prpBCDE
operon is ensured by the Pdu protein that has CobB-like activity. Since synthesis of this Pdu protein depends on the availability of 1,2-propanediol, the cell solves the problem faced in an environment devoid of 1,2-propanediol where propionate is the sole carbon and energy source by having
cobB
located outside of the
pdu
operon and its expression independent of 1,2-propanediol. At present, it is unclear how the CobB and Pdu proteins affect
prpBCDE
expression.
A Putative Multisubunit Na
+
/H
+
Antiporter from
Staphylococcus aureus
Authors: Hiramatsu Toshiaki , Kodama Kazuyo , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: December 2019

We cloned several genes encoding an Na
+
/H
+
antiporter of
Staphylococcus aureus
from chromosomal DNA by using an
Escherichia coli
mutant, lacking all of the major Na
+
/H
+
antiporters, as the host.
E. coli
cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na
+
/H
+
antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na
+
/H
+
antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na
+
/H
+
antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na
+
/H
+
antiporter. We observed a large Na
+
extrusion activity, driven by respiration in
E. coli
cells harboring the plasmid carrying the genes. The Na
+
extrusion was sensitive to an H
+
conductor, supporting the idea that the system is not a respiratory Na
+
pump but an Na
+
/H
+
antiporter. Introduction of the plasmid into
E. coli
mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.
Expression of the
Bacillus subtilis acsA
Gene: Position and Sequence Context Affect
cre
-Mediated Carbon Catabolite Repression
Authors: Zalieckas Jill M. , Wray Lewis V. , Fisher Susan H.
Published: December 2019

In
Bacillus subtilis
, carbon catabolite repression (CCR) of many genes is mediated at
cis
-acting carbon repression elements (
cre
) by the catabolite repressor protein CcpA. Mutations in transcription-repair coupling factor (
mfd
) partially relieve CCR at
cre
sites located downstream of transcriptional start sites by abolishing the Mfd-mediated displacement of RNA polymerase stalled at
cre
sites which act as transcriptional roadblocks. Although the
acsA cre
is centered 44.5 bp downstream of the
acsA
transcriptional start site, CCR of
acsA
expression is not affected by an
mfd
mutation. When the
acsA cre
is centered 161.5 bp downstream of the transcriptional start site for the unregulated
tms
promoter, CCR is partially relieved by the
mfd
mutation. Since CCR mediated at an
acsA cre
centered 44.5 bp downstream of the
tms
start site is not affected by the
mfd
mutation, the inability of Mfd to modulate CCR of
acsA
expression most likely results from the location of the
acsA cre
. Higher levels of CCR were found to occur at
cre
sites flanked by A+T-rich sequences than at
cre
sites bordered by G and C nucleotides. This suggests that nucleotides adjacent to the proposed 14-bp
cre
consensus sequence participate in the formation of the CcpA catabolite repression complex at
cre
sites. Examination of CCR of
acsA
expression revealed that this regulation required the Crh and seryl-phosphorylated form of the HPr proteins but not glucose kinase.
Functional Similarities between the
Listeria monocytogenes
Virulence Regulator PrfA and Cyclic AMP Receptor Protein: the PrfA* (Gly145Ser) Mutation Increases Binding Affinity for Target DNA
Authors: Vega Yolanda , Dickneite Carmen , Ripio María-Teresa , Böckmann Regine , González-Zorn Bruno , Novella Susana , Domínguez-Bernal Gustavo , Goebel Werner , Vázquez-Boland José A.
Published: December 2019

Most
Listeria monocytogenes
virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP). Its coding gene,
prfA
, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent
plcA
promoter. We have recently characterized
prfA
* mutants from
L. monocytogenes
which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress
prfA
and the genes of the PrfA virulence regulon. Here, we show that about 10 times more PrfA protein is produced in a
prfA
* strain than in the wild type. Thus, the phenotype of
prfA
* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback. We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA. Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the
plcA
promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes. In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA*. These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence. Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP. Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor.
The Modified β-Ketoadipate Pathway in
Rhodococcus rhodochrous
N75: Enzymology of 3-Methylmuconolactone Metabolism
Authors: Cha Chang-Jun , Cain Ronald B. , Bruce Neil C.
Published: December 2019

Rhodococcus rhodochrous
N75 is able to metabolize 4-methylcatechol via a modified β-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of
R. rhodochrous
N75 on
p
-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native
M
r
of 128,000, and a subunit
M
r
of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.
New Small, Acid-Soluble Proteins Unique to Spores of
Bacillus subtilis
: Identification of the Coding Genes and Regulation and Function of Two of These Genes
Authors: Bagyan Irina , Setlow Barbara , Setlow Peter
Published: December 2019

Eleven small, acid-soluble proteins (SASP) which are present in spores but not in growing cells of
Bacillus subtilis
were identified by sequence analysis of proteins separated by acrylamide gel electrophoresis of acid extracts from spores which lack the three major SASP (α, β, and γ). Six of these proteins are encoded by open reading frames identified previously or by analysis of the complete sequence of the
B. subtilis
genome, including two minor α/β-type SASP (SspC and SspD) and a putative spore coat protein (CotK). Five proteins are encoded by short open reading frames that were not identified as coding regions in the analysis of the complete
B. subtilis
genomic sequence. Studies of the regulation of two of the latter genes, termed
sspG
and
sspJ
, showed that both are expressed only in sporulation. The
sspG
gene is transcribed in the mother cell compartment by RNA polymerase with the mother cell-specific sigma factor for RNA polymerase, ς
K
, and is cotranscribed with a downstream gene,
yurS
;
sspG
transcription also requires the DNA binding protein GerE. In contrast,
sspJ
is transcribed in the forespore compartment by RNA polymerase with the forespore-specific ς
G
and appears to give a monocistronic transcript. A mutation eliminating SspG had no effect on sporulation or spore properties, while loss of SspJ caused a slight decrease in the rate of spore outgrowth in an otherwise wild-type background.
Insertion Mutagenesis of the Ferric Pyoverdine Receptor FpvA of
Pseudomonas aeruginosa
: Identification of Permissive Sites and a Region Important for Ligand Binding
Authors: Kilburn Laurie , Poole Keith , Meyer Jean-Marie , Neshat Shadi
Published: December 2019

Insertion of an 18-amino-acid-encoding sequence within the
fpvA
gene identified permissive sites at residues Y350, A402, R451, R521, and R558, consistent with these residues occurring in extramembranous loop regions of the protein. Insertions at R451, R521, and R558 did not adversely affect receptor function, although insertions at Y350 and A402 compromised ferric pyoverdine binding and uptake. The latter region likely contributes to or interacts with the ligand-binding site.
Nitrite-Specific Active Transport System of the Cyanobacterium
Synechococcus
sp. Strain PCC 7942
Authors: Maeda Shin-ichi , Okamura Masato , Kobayashi Masaki , Omata Tatsuo
Published: December 2019

Studies on the nitrite uptake capability of a mutant of
Synechococcus
sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent

K
m

(NO
2
−
) of about 20 μM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium.