bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Methylation Sites and Effects of Phototaxis Stimuli on Transducer Methylation in
Halobacterium salinarum
Authors: Perazzona Bastianella , Spudich John L.
Published: December 2019

The two transducers in the phototaxis system of the archaeon
Halobacterium salinarum
, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo
l
-[
methyl
-
3
H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type
H. salinarum
cells. This result is unlike that obtained with
Escherichia coli
, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with
Bacillus subtilis
, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of
H. salinarum
exhibited the
E. coli
-like asymmetric pattern, as has recently also been observed in
B. subtilis
. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.
Role of the Alkyl Hydroperoxide Reductase (
ahpCF
) Gene in Oxidative Stress Defense of the Obligate Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Smith C. Jeffrey
Published: December 2019

In this study we report the identification and role of the alkyl hydroperoxide reductase (
ahp
) gene in
Bacteroides fragilis
. The two components of
ahp
,
ahpC
, and
ahpF
, are organized in an operon, and the deduced amino acid sequences revealed that
B. fragilis
AhpCF shares approximately 60% identity to orthologues in other gram-positive and gram-negative bacteria. Northern blot hybridization analysis of total RNA showed that the
ahpCF
genes were transcribed as a polycistronic 2.4-kb mRNA and that
ahpC
also was present as a 0.6-kb monocistronic mRNA.
ahpC
and
ahpCF
mRNAs were induced approximately 60-fold following H
2
O
2
treatment or oxygen exposure of the parent strain but were constitutive in a peroxide-resistant strain. Further investigation using an
ahpCF
′::β-xylosidase gene transcriptional fusion confirmed that
ahpCF
had lost normal regulation in the peroxide-resistant strain compared to the parent. The
ahpCF
mutant was more sensitive to growth inhibition and mutagenesis by organic peroxides than the parent strain, as determined by disk inhibition assays and the frequency of mutation to fusidic acid resistance. This finding suggests that the
ahp
genes play an important role in peroxide resistance in
B. fragilis
. Under anaerobic conditions, we observed increases in the number of spontaneous fusidic acid-resistant mutants of five- and sevenfold in
ahpCF
and
ahpF
strain backgrounds, respectively, and eightfold in the
ahpCF katB
double mutant strain compared to the parent and
katB
strains. In addition,
ahpCF
,
ahpF
, and
ahpCF katB
mutants were slightly more sensitive to oxygen exposure than the parent strain. Moreover, the isolation of a strain with enhanced aerotolerance and high-level resistance to alkyl hydroperoxides from an
ahpCF katB
parent suggests that the physiological responses to peroxide toxicity and to the toxic effects of molecular oxygen are overlapping and complex in this obligate anaerobe.
The
vsp
Locus of
Mycoplasma bovis
: Gene Organization and Structural Features
Authors: Lysnyansky Inessa , Sachse Konrad , Rosenbusch Ricardo , Levisohn Sharon , Yogev David
Published: December 2019

Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen
Mycoplasma bovis
were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the
M. bovis
chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395–5401, 1996). In the present study, 13 single-copy
vsp
genes organized in a chromosomal cluster were identified and characterized. All
vsp
genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each
vsp
coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various
vsp
genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each
vsp
gene is also 5′ linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the
vsp
genes and within the
vsp
coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the
vsp
locus of
M. bovis
.
Characterization ofEnterococcus faecalisAlkaline Phosphatase and Use in IdentifyingStreptococcus agalactiaeSecreted Proteins
Authors: Lee Martin H. , Nittayajarn Aphakorn , Ross R. Paul , Rothschild Cynthia B. , Parsonage Derek , Claiborne Al , Rubens Craig E.
Published: December 2019

We have identified and characterized anEnterococcus faecalisalkaline phosphatase (AP, encoded byphoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases fromBacillus subtilis. Expression ofphoZinEscherichia coli,E. faecalis,Streptococcus agalactiae(group B streptococcus [GBS]), orStreptococcus pyogenes(group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme’s subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). InE. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant,phoZΔss, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, andE. faecalisand is expected to be useful in a variety of gram-positive bacteria.
Essential Role of the Iron-Regulated Outer Membrane Receptor FauA in Alcaligin Siderophore-Mediated Iron Uptake in
Bordetella
Species
Authors: Brickman Timothy J. , Armstrong Sandra K.
Published: December 2019

Phenotypic analysis using heterologous host systems localized putative
Bordetella pertussis
ferric alcaligin transport genes and Fur-binding sequences to a 3.8-kb genetic region downstream from the
alcR
regulator gene. Nucleotide sequencing identified a TonB-dependent receptor family homolog gene,
fauA
, predicted to encode a polypeptide with high amino acid sequence similarity with known bacterial ferric siderophore receptors. In
Escherichia coli
, the
fauA
genes of both
B. pertussis
and
Bordetella bronchiseptica
directed the production of a 79-kDa polypeptide, approximating the predicted size of the mature FauA protein.
B. bronchiseptica fauA
insertion mutant BRM17 was unable to utilize ferric alcaligin, and in complementation analyses ferric alcaligin utilization was restored to this mutant by supplying the wild-type
fauA
gene in
trans
. Mutant BRM18, carrying a nonpolar in-frame
fauA
deletion mutation, was defective in ferric alcaligin utilization and
55
Fe-ferric alcaligin uptake and no longer produced a 79-kDa iron-regulated outer membrane protein. In complementation analyses, BRM18 merodiploids bearing the wild-type
fauA
gene in
trans
regained ferric alcaligin siderophore transport and utilization functions and produced the 79-kDa protein. Analysis of a plasmid-borne
fauA-lacZ
operon fusion confirmed that
fauA
is subject to iron regulation at the transcriptional level and that
cis
-acting transcriptional control elements mediating
fauA
iron repressibility reside within the 3.8-kb
Pst
I
fauA
DNA region. Moreover, expression of the
fauA-lacZ
fusion gene under iron starvation conditions was shown to be
alcR
dependent. FauA is a 79-kDa iron-regulated outer membrane receptor protein required for transport and utilization of ferric alcaligin siderophore complexes by
Bordetella
species.
Abiotic Surface Sensing and Biofilm-Dependent Regulation of Gene Expression in
Escherichia coli
Authors: Prigent-Combaret Claire , Vidal Olivier , Dorel Corinne , Lejeune Philippe
Published: December 2019

To get further information on bacterial surface sensing and biofilm-dependent regulation of gene expression in
Escherichia coli
K-12, random insertion mutagenesis with Mu dX, a mini-Mu carrying the promoterless
lacZ
gene, was performed with an
ompR234
adherent strain, and a simple screen was developed to assess changes in gene expression in biofilm cells versus planktonic cells. This screen revealed that major changes in the pattern of gene expression occur during biofilm development: the transcription of 38% of the genes was affected within biofilms. Different cell functions were more expressed in sessile bacteria: the OmpC porin, the high-affinity transport system of glycine betaine (encoded by the
proU
operon), the colanic acid exopolysaccharide (
wca
locus, formerly called
cps
), tripeptidase T (
pepT
), and the nickel high-affinity transport system (
nikA
). On the other hand, the syntheses of flagellin (
fliC
) and of a putative protein of 92 amino acids (
f92
) were both reduced in biofilms. Such a genetic reprogramming of gene expression in biofilms seems to result from changes in multiple environmental physicochemical conditions. In this work, we show that bacteria within biofilms encounter higher-osmolarity conditions, greater oxygen limitation, and higher cell density than in the liquid phase.
A Mutant HemA Protein with Positive Charge Close to the N Terminus Is Stabilized against Heme-Regulated Proteolysis in
Salmonella typhimurium
Authors: Wang Liying , Wilson Sandra , Elliott Thomas
Published: December 2019

The HemA enzyme (glutamyl-tRNA reductase) catalyzes the first committed step in heme biosynthesis in the enteric bacteria. HemA is mainly regulated by conditional protein stability; it is stable and, consequently, more abundant in heme-limited cells but unstable and less abundant in normally growing cells. Both the Lon and ClpAP energy-dependent proteases contribute to HemA turnover in vivo. Here we report that the addition of two positively charged lysine residues to the third and fourth positions at the HemA N terminus resulted in complete stabilization of the protein. By contrast, the addition of an N-terminal myc epitope tag did not affect turnover. This result confirms the importance of the N-terminal sequence for proteolysis of HemA. This region of the protein also contains a proline flanked by hydrophobic residues, a motif that has been suggested to be important for Lon-mediated proteolysis of UmuD. However, mutation of this motif did not affect the turnover of HemA protein. Cells expressing the stabilized HemA[KK] mutant protein display substantial defects in heme regulation.
rsmC
of the Soft-Rotting Bacterium
Erwinia carotovora
subsp.
carotovora
Negatively Controls Extracellular Enzyme and Harpin
Ecc
Production and Virulence by Modulating Levels of Regulatory RNA (
rsmB
) and RNA-Binding Protein (RsmA)
Authors: Cui Yaya , Mukherjee Asita , Dumenyo C. Korsi , Liu Yang , Chatterjee Arun K.
Published: December 2019

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin
Ecc
(the elicitor of hypersensitive reaction) in
Erwinia carotovora
subsp.
carotovora
is regulated by RsmA, an RNA-binding protein, and
rsmB
, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219–234, 1998). We have cloned and characterized a novel regulatory gene,
rsmC
, that activates RsmA production and represses extracellular enzyme and harpin
Ecc
production,
rsmB
transcription, and virulence in
E. carotovora
subsp.
carotovora
. In an
rsmC
knockout mutant of
E. carotovora
subsp.
carotovora
Ecc71 carrying the chromosomal copy of the wild-type
rsmA
+
allele, the basal levels of Pel, Peh, Cel, Prt, and harpin
Ecc
as well as the amounts of
rsmB
,
pel-1
,
peh-1
,
celV
, and

hrpN
Ecc

transcripts are high, whereas the levels of
rsmA
transcripts and RsmA protein are low. Furthermore, the expression of an
rsmA-lacZ
gene fusion is lower in the RsmC
−
mutant than in the RsmC
+
parent. Conversely, the expression of an
rsmB-lacZ
operon fusion is higher in the RsmC
−
mutant than in the RsmC
+
parent. These observations establish that RsmC negatively regulates
rsmB
transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC
−
mutant, an RsmA
−
mutant, and an RsmA
−
RsmC
−
double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on
rsmA
and
rsmB
. Exoprotein production by the RsmC
−
mutant is partially dependent on the quorum sensing signal,
N
-(3-oxohexanoyl)-
l
-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of
rsmC
sequences in
E. carotovora
subsp.
atroseptica
,
E. carotovora
subsp.
betavasculorum
, and
E. carotovora
subsp.
carotovora
. These findings collectively support the idea that
rsmA
and
rsmB
expression in these plant pathogenic
Erwinia
species is controlled by RsmC or a functional homolog of RsmC.
Sequence Analysis of the Gene Encoding Amylosucrase from
Neisseria polysaccharea
and Characterization of the Recombinant Enzyme
Authors: Potocki De Montalk G. , Remaud-Simeon M. , Willemot R. M. , Planchot V. , Monsan P.
Published: December 2019

The
Neisseria polysaccharea
gene encoding amylosucrase was subcloned and expressed in
Escherichia coli
. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)
8
-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione
S
-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.
Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination
Authors: Peña Carol E. A. , Kahlenberg J. Michelle , Hatfull Graham F.
Published: December 2019

The temperate mycobacteriophage L5 integrates site specifically into the genomes of
Mycobacterium smegmatis
,
Mycobacterium tuberculosis
, and
Mycobacterium bovis
bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5
attP
site and the mycobacterial
attB
site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that
attP
, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of
attB
capture and formation of products. The arm-type integrase binding sites within
attP
play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the
attP
core, while an additional pair of sites, P1-P2, is required for interaction with
attB
.
Slipped Misalignment Mechanisms of Deletion Formation: In Vivo Susceptibility to Nucleases
Authors: Bzymek Malgorzata , Saveson Catherine J. , Feschenko Vladimir V. , Lovett Susan T.
Published: December 2019

Misalignment of repeated sequences during DNA replication can lead to deletions or duplications in genomic DNA. In
Escherichia coli
, such genetic rearrangements can occur at high frequencies, independent of the RecA-homologous recombination protein, and are sometimes associated with sister chromosome exchange (SCE). Two mechanisms for RecA-independent genetic rearrangements have been proposed: simple replication misalignment of the nascent strand and its template and SCE-associated misalignment involving both nascent strands. We examined the influence of the 3′ exonuclease of DNA polymerase III and exonuclease I on deletion via these mechanisms in vivo. Because mutations in these exonucleases stimulate tandem repeat deletion, we conclude that displaced 3′ ends are a common intermediate in both mechanisms of slipped misalignments. Our results also confirm the notion that two distinct mechanisms contribute to slipped misalignments: simple replication misalignment events are sensitive to DNA polymerase III exonuclease, whereas SCE-associated events are sensitive to exonuclease I. If heterologies are present between repeated sequences, the mismatch repair system dependent on MutS and MutH aborts potential deletion events via both mechanisms. Our results suggest that simple slipped misalignment and SCE-associated misalignment intermediates are similarly susceptible to destruction by the mismatch repair system.
Temporal Expression of the
Bacillus subtilis secA
Gene, Encoding a Central Component of the Preprotein Translocase
Authors: Herbort Markus , Klein Michael , Manting Erik H. , Driessen Arnold J. M. , Freudl Roland
Published: December 2019

In
Bacillus subtilis
, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the
secA
gene, encoding a central component of the
B. subtilis
preprotein translocase. We found that
secA
and the downstream gene (
prfB
) constitute an operon that is transcribed from a vegetative (ς
A
-dependent) promoter located upstream of
secA
. Furthermore, using different independent methods, we found that
secA
expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of
secA
sharply decreased to a low basal level. Since at the time of maximal
secA
transcription the secretion activity of
B. subtilis
strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the
B. subtilis
protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed.
Localization of FtsI (PBP3) to the Septal Ring Requires Its Membrane Anchor, the Z Ring, FtsA, FtsQ, and FtsL
Authors: Weiss David S. , Chen Joseph C. , Ghigo Jean-Marc , Boyd Dana , Beckwith Jon
Published: December 2019

Assembly of the division septum in bacteria is mediated by several proteins that localize to the division site. One of these, FtsI (also called penicillin-binding protein 3) of
Escherichia coli
, consists of a short cytoplasmic domain, a single membrane-spanning segment, and a large periplasmic domain that encodes a transpeptidase activity involved in synthesis of septal peptidoglycan. We have constructed a merodiploid strain with a wild-type copy of
ftsI
at the normal chromosomal locus and a genetic fusion of
ftsI
to the green fluorescent protein (
gfp
) at the lambda attachment site.
gfp-ftsI
was expressed at physiologically appropriate levels under control of a regulatable promoter. Consistent with previous results based on immunofluorescence microscopy GFP-FtsI localized to the division site during the later stages of cell growth and throughout septation. Localization of GFP-FtsI to the cell pole(s) was not observed unless the protein was overproduced about 10-fold. Membrane anchor alterations shown previously to impair division but not membrane insertion or transpeptidase activity were found to interfere with localization of GFP-FtsI to the division site. In contrast, GFP-FtsI localized well in the presence of β-lactam antibiotics that inhibit the transpeptidase activity of FtsI. Septal localization depended upon every other division protein tested (FtsZ, FtsA, FtsQ, and FtsL). We conclude that FtsI is a late recruit to the division site, and that its localization depends on an intact membrane anchor.
Motility of
Helicobacter pylori
Is Coordinately Regulated by the Transcriptional Activator FlgR, an NtrC Homolog
Authors: Spohn Gunther , Scarlato Vincenzo
Published: December 2019

ς
54
is the subunit of bacterial RNA polymerase that transcribes from promoters with enhancer elements bound by enhancer-binding proteins. By computer searches of
Helicobacter pylori
genomic sequences, chromosomal gene disruption, and RNA analyses, we have identified ς
54
-recognized promoters that regulate transcription of flagellar basal body and hook genes, as well as the enhancer-binding protein FlgR (flagellum regulator), a transactivating protein of the NtrC family. We demonstrate that FlgR is required for bacterial motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR acts as a repressor of transcription of the ς
28
-regulated
flaA
flagellin gene promoter, while changes in DNA topology repress transcription of the ς
54
-regulated
flaB
flagellin gene promoter. Our data indicate that regulation of flagellar gene expression in
H. pylori
shows similarities with that in enterobacteriaceae and
Caulobacter
.
Role of Glycosylation at Ser63 in Production of Soluble Pilin in Pathogenic
Neisseria
Authors: Marceau Michael , Nassif Xavier
Published: December 2019

Pilus-mediated adhesion is essential in the pathogenesis of
Neisseria meningitidis
(MC) and
Neisseria gonorrhoeae
(GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.
Identification of SycN, YscX, and YscY, Three New Elements of the
Yersinia
Yop Virulon
Authors: Iriarte Maite , Cornelis Guy R.
Published: December 2019

The Yop virulon allows
Yersinia
spp. to resist the immune response of the host by injecting harmful proteins into host cells. We identified three new elements of the Yop virulon: SycN, required for normal secretion of YopN, and YscX and YscY, two new components of the secretion machinery.
Control of
Herbaspirillum seropedicae
NifA Activity by Ammonium Ions and Oxygen
Authors: Souza E. M. , Pedrosa F. O. , Drummond M. , Rigo L. U. , Yates M. G.
Published: December 2019

The activity of a truncated form of
Herbaspirillum seropedicae
NifA in different genetic backgrounds showed that its regulatory domain is involved in nitrogen control but not in O
2
sensitivity or Fe dependence. The model for nitrogen control involving PII could thus apply to the proteobacteria at large. NifA may have a role in controlling ADP-ribosylation of nitrogenase in
Azospirillum brasilense
.
Regulation of the Transposase of Tn
4652
by the Transposon-Encoded Protein TnpC
Authors: Hõrak Rita , Kivisaar Maia
Published: December 2019

Transposition is a DNA reorganization reaction potentially deleterious for the host. The frequency of transposition is limited by the amount of transposase. Therefore, strict regulation of a transposase is required to keep control over the destructive multiplication of the mobile element. We have shown previously that the expression of the transposase (
tnpA
) of the
Pseudomonas putida
PaW85 transposon Tn
4652
is positively affected by integration host factor. Here, we present evidence that the amount of the transposase of Tn
4652
in
P. putida
cells is controlled by the transposon-encoded protein (TnpC). Sequence analysis of the 120-amino-acid-long TnpC, coded just downstream of the
tnpA
gene, showed that it has remarkable similarity to the putative polypeptide encoded by the mercury resistance transposon Tn
5041
. As determined by quantitative Western blot analysis, the abundance of TnpA was reduced up to 10-fold in the intact
tnpC
background. In vivo experiments using transcriptional and translational fusions of the
tnpA
gene and the reporter gene
gusA
indicated that TnpC operates in the regulation of the transposase of Tn
4652
at the post-transcriptional level.
MinDE-Dependent Pole-to-Pole Oscillation of Division Inhibitor MinC in
Escherichia coli
Authors: Raskin David M. , de Boer Piet A. J.
Published: December 2019

By inhibiting FtsZ ring formation near the cell ends, the MinC protein plays a critical role in proper positioning of the division apparatus in
Escherichia coli
. MinC activity requires that of MinD, and the MinE peptide provides topological specificity by suppressing MinC-MinD-mediated division inhibition specifically at the middle of the cell. We recently presented evidence that MinE not only accumulates in an FtsZ-independent ring structure at the cell’s middle but also imposes a unique dynamic localization pattern upon MinD in which the latter accumulates alternately in either one of the cell halves in what appears to be a rapidly oscillating membrane association-dissociation cycle. Here we show that functional green fluorescent protein-MinC displays a very similar oscillatory behavior which is dependent on both MinD and MinE and independent of FtsZ. The results support a model in which MinD recruits MinC to its site of action and in which FtsZ ring assembly at each of the cell ends is blocked in an intermittent and alternate fashion.
Functional Genomics: Expression Analysis of
Escherichia coli
Growing on Minimal and Rich Media
Authors: Tao Han , Bausch Christoph , Richmond Craig , Blattner Frederick R. , Conway Tyrrell
Published: December 2019

DNA arrays of the entire set of
Escherichia coli
genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose. Ratios of the transcript levels for all 4,290
E. coli
protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained. The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells. The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways. Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and
rpoS
expression was similarly elevated. The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced. The hallmark features of
E. coli
cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress. A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated. This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest.
Identification and Characterization of Major Lipid Particle Proteins of the Yeast
Saccharomyces cerevisiae
Authors: Athenstaedt Karin , Zweytick Dagmar , Jandrositz Anita , Kohlwein Sepp Dieter , Daum Günther
Published: December 2019

Lipid particles of the yeast
Saccharomyces cerevisiae
were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.
Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases
Authors: D’Argenio David A. , Vetting Mathew W. , Ohlendorf Douglas H. , Ornston L. Nicholas
Published: December 2019

Protocatechuate 3,4-dioxygenase is a member of a family of bacterial enzymes that cleave the aromatic rings of their substrates between two adjacent hydroxyl groups, a key reaction in microbial metabolism of varied environmental chemicals. In an appropriate genetic background, it is possible to select for
Acinetobacter
strains containing spontaneous mutations blocking expression of
pcaH
or -
G
, genes encoding the α and β subunits of protocatechuate 3,4-dioxygenase. The crystal structure of the
Acinetobacter
oxygenase has been determined, and this knowledge affords us the opportunity to understand how mutations alter function in the enzyme. An earlier investigation had shown that a large fraction of spontaneous mutations inactivating
Acinetobacter
protocatechuate oxygenase are either insertions or large deletions. Therefore, the prior procedure of mutant selection was modified to isolate
Acinetobacter
strains in which mutations within
pcaH
or -
G
cause a heat-sensitive phenotype. These mutations affected residues distributed throughout the linear amino acid sequences of PcaH and PcaG and impaired the dioxygenase to various degrees. Four of 16 mutants had insertions or deletions in the enzyme ranging in size from 1 to 10 amino acid residues, highlighting areas of the protein where large structural changes can be tolerated. To further understand how protein structure influences function, we isolated strains in which the phenotypes of three different deletion mutations in
pcaH
or -
G
were suppressed either by a spontaneous mutation or by a PCR-generated random mutation introduced into the
Acinetobacter
chromosome by natural transformation. The latter procedure was also used to identify a single amino acid substitution in PcaG that conferred activity towards catechol sufficient for growth with benzoate in a strain in which catechol 1,2-dioxygenase was inactivated.
Horizontal Transfer of Genetic Material among
Saccharomyces
Yeasts
Authors: Marinoni Gaelle , Manuel Martine , Petersen Randi Føns , Hvidtfeldt Jeanne , Sulo Pavol , Piškur Jure
Published: December 2019

The genus
Saccharomyces
consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different
Saccharomyces
species. All of these strains secreted an a- or α-like pheromone recognized by
S. cerevisiae
tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that
Saccharomyces
yeasts have a potential to exchange genetic material. If
Saccharomyces
isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.
Divergent Regulation of the Evolutionarily Closely Related Promoters of the
Saccharomyces cerevisiae STA2
and
MUC1
Genes
Authors: Gagiano Marco , Van Dyk Dewald , Bauer Florian F. , Lambrechts Marius G. , Pretorius Isak S.
Published: December 2019

The 5′ upstream regions of the
Saccharomyces cerevisiae
glucoamylase-encoding genes
STA1
to -
3
and of the
MUC1
(or
FLO11
) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent. Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view. Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level. To investigate the transcriptional regulation, we compared the transcription levels conferred by the
STA2
and
MUC1
promoters under various growth conditions. Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects. We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression. We also identified the
STA10
repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in
S. cerevisiae.
Genetic Analysis of
nif
Regulatory Genes by Utilizing the Yeast Two-Hybrid System Detected Formation of a NifL-NifA Complex That Is Implicated in Regulated Expression of
nif
Genes
Authors: Lei Shi , Pulakat Lakshmidevi , Gavini Narasaiah
Published: December 2019

In diazotrophic organisms, nitrogenase synthesis and activity are tightly regulated. Two genes,
nifL
and
nifA
, are implicated as playing a major role in this regulation. NifA is a transcriptional activator, and its activity is inhibited by NifL in response to availability of excess fixed nitrogen and high O
2
tension. It was postulated that NifL binds to NifA to inhibit NifA-mediated transcriptional activation of
nif
genes. Mutational analysis combined with transcriptional activation studies clearly is in agreement with the proposal that NifL interacts with NifA. However, several attempts to identify NifA-NifL interactions by using methods such as coimmunoprecipitations and chemical cross-linking experiments failed to detect direct interactions between these proteins. Here we have taken a genetic approach, the use of a yeast two-hybrid protein-protein interaction assay system, to investigate NifL interaction with NifA. A DNA fragment corresponding to the kinase-like domain of
nifL
was PCR amplified and was used to generate translation fusions with the DNA binding domain and the DNA activation domain of the yeast transcriptional activator GAL4 in yeast two-hybrid vectors. Similarly, a DNA fragment corresponding to the catalytic domain of
nifA
was PCR amplified and used to generate translation fusions with the DNA-binding domain and the DNA-activation domain of GAL4 in yeast two-hybrid vectors. After introducing appropriate plasmid combinations in yeast cells, the existance of direct interaction between NifA and NifL was analyzed with the MATCHMAKER yeast two-hybrid system by testing for the expression of
lacZ
and
his3
genes. These analyses showed that the kinase-like domain of NifL directly interacts with the catalytic domain of NifA.
Porins in the Cell Wall of
Mycobacterium tuberculosis
Authors: Kartmann Bettina , Stengler Steffen , Niederweis Michael
Published: December 2019

Lipid bilayer experiments indicated that the cell wall of
Mycobacterium tuberculosis
contains at least two different porins: (i) a cation-selective, heat-sensitive 0.7-nS channel which has a short-lived open state and is probably composed of 15-kDa subunits and (ii) a 3-nS, >60-kDa channel with a long-lived open state, resembling porins from fast-growing mycobacteria.
Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in
Streptococcus pneumoniae
Authors: Hoskins JoAnn , Matsushima Patti , Mullen Deborah L. , Tang Joseph , Zhao Genshi , Meier Timothy I. , Nicas Thalia I. , Jaskunas S. Richard
Published: December 2019

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in
Streptococcus pneumoniae
were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a
pbp1a pbp1b
double-disruption mutant grew more slowly than its parent did. Attempts to generate a
pbp1a pbp2a
double-disruption mutant failed. The
pbp2a
mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the
pbp1a
,
pbp1b
, and
pbp2a
genes are dispensable but that either
pbp1a
or
pbp2a
is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in
S. pneumoniae
.
Occurrence of Free
d
-Amino Acids and Aspartate Racemases in Hyperthermophilic Archaea
Authors: Matsumoto Megumi , Homma Hiroshi , Long Zhiqun , Imai Kazuhiro , Iida Toshii , Maruyama Tadashi , Aikawa Yuko , Endo Isao , Yohda Masafumi
Published: December 2019

The occurrence of free
d
-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of
d
-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70°C, and aspartate racemase homologous genes in them were identified by PCR.
d
-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in
Thermococcus
strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that
d
-amino acids are produced in some hyperthermophilic archaea, although their function is unknown.
The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by
o349
of the
wec
Gene Cluster of
Escherichia coli
K-12
Authors: Barr Kathleen , Klena John , Rick Paul D.
Published: December 2019

The assembly of the phosphoglyceride-linked form of enterobacterial common antigen (ECA
PG
) occurs by a mechanism that involves modulation of polysaccharide chain length. However, the genetic determinant of this modulation has not been identified. Site-directed mutagenesis of
o349
of the
Escherichia coli
K-12
wec
gene cluster revealed that this locus encodes a Wzz protein that specifically modulates the chain length of ECA
PG
polysaccharides, and we have designated this locus
wzz
ECA
. The Wzz
ECA
-mediated modulation of ECA
PG
polysaccharide chains is the first demonstrated example of Wzz regulation involving a polysaccharide that is not linked to the core-lipid A structure of lipopolysaccharide.
On the Origin of Branches in
Escherichia coli
Authors: Gullbrand Björn , Åkerlund Thomas , Nordström Kurt
Published: December 2019

Some
Escherichia coli
strains with impaired cell division form branched cells at high frequencies during certain growth conditions. Here, we show that neither FtsI nor FtsZ activity is required for the development of branches. Buds did not form at specific positions along the cell surface during high-branching conditions. Antibiotics affecting cell wall synthesis had a positive effect on branch formation in the case of ampicillin, cephalexin, and penicillin G, whereas mecillinam and
d
-cycloserine had no substantial effect. Altering the cell morphology by nutritional shifts showed that changes in morphology preceded branching, indicating that the cell’s physiological state rather than specific medium components induced branching. Finally, there was no increased probability for bud formation in the daughters of a cell with a bud or branch, showing that bud formation is a random event. We suggest that branch formation is caused by abnormalities in cell wall elongation rather than by aberrant cell division events.
Characterization of a Hank’s Type Serine/Threonine Kinase and Serine/Threonine Phosphoprotein Phosphatase in
Pseudomonas aeruginosa
Authors: Mukhopadhyay Subhendu , Kapatral Vinayak , Xu Wenbin , Chakrabarty A. M.
Published: December 2019

Pseudomonas aeruginosa
is an opportunistic pathogen that causes infections in eye, urinary tract, burn, and immunocompromised patients. We have cloned and characterized a serine/threonine (Ser/Thr) kinase and its cognate phosphoprotein phosphatase. By using oligonucleotides from the conserved regions of Ser/Thr kinases of mycobacteria, an 800-bp probe was used to screen
P. aeruginosa
PAO1 genomic library. A 20-kb cosmid clone was isolated, from which a 4.5-kb DNA with two open reading frames (ORFs) were subcloned. ORF1 was shown to encode Ser/Thr phosphatase (Stp1), which belongs to the PP2C family of phosphatases. Overlapping with the
stp1
ORF, an ORF encoding Hank’s type Ser/Thr kinase was identified. Both ORFs were cloned in pGEX-4T1 and expressed in
Escherichia coli
. The overexpressed proteins were purified by glutathione-Sepharose 4B affinity chromatography and were biochemically characterized. The Stk1 kinase is 39 kDa and undergoes autophosphorylation and can phosphorylate eukaryotic histone H1. A site-directed Stk1 (K86A) mutant was shown to be incapable of autophosphorylation. A two-dimensional phosphoamino acid analysis of Stk1 revealed strong phosphorylation at a threonine residue and weak phosphorylation at a serine residue. The Stp1 phosphatase is 27 kDa and is an Mn
2+
-, but not a Ca
2+
- or a Mg
2+
-, dependent Ser/Thr phosphatase. Its activity is inhibited by EDTA and NaF, but not by okadaic acid, and is similar to that of PP2C phosphatase.