bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Activation of the Chlorocatechol Degradative Genes of
Ralstonia eutropha
NH9
Authors: Ogawa Naoto , McFall Sally M. , Klem Thomas J. , Miyashita Kiyotaka , Chakrabarty A. M.
Published: December 2019

Ralstonia eutropha
(formerly
Alcaligenes eutrophus
) NH9 degrades 3-chlorobenzoate via the modified
ortho
-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the
cbnABCD
operon encoding the degradative enzymes (including
orfX
of unknown function) and the divergently transcribed
cbnR
gene encoding the LysR-type transcriptional regulator of the
cbn
operon. The
cbnRAB orfXCD
gene cluster is nearly identical to the chlorocatechol genes (
tcbRCD orfXEF
) of the 1,2,4-trichlorobenzene-degrading bacterium
Pseudomonas
sp. strain P51. Transcriptional fusion studies demonstrated that
cbnR
regulates the expression of
cbnABCD
positively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-
cis
,
cis
-muconate (2-CM) and
cis
,
cis
-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (
catBCA
) and chlorocatechol (
clcABD
) operons of
Pseudomonas putida
, in which only the intermediates of the regulated pathway, CCM for
catBCA
and 2-CM for
clcABD
, act as significant inducers. Specific binding of CbnR protein to the
cbnA
promoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position −20 to position −80 upstream of the
cbnA
transcriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position −50. Circular permutation gel shift assays demonstrated that CbnR bent the
cbnA
promoter region to an angle of 78° and that this angle was relaxed to 54° upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the
catBCA
and
clcABD
promoters may indicate a conserved transcriptional activation mechanism of
ortho
-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.
A Novel Cellulosomal Scaffoldin from
Acetivibrio cellulolyticus
That Contains a Family 9 Glycosyl Hydrolase
Authors: Ding Shi-You , Bayer Edward A. , Steiner David , Shoham Yuval , Lamed Raphael
Published: December 2019

A novel cellulosomal scaffoldin gene, termed
cipV
, was identified and sequenced from the mesophilic cellulolytic anaerobe
Acetivibrio cellulolyticus
. Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of
Clostridium thermocellum
. The
cipV
gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496. CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus. Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases. Sequence analysis indicated the following. (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins. (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins. (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted “recognition codes” differ from those proposed for the other known dockerins. The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins. (iv) The X2 module has about 60% sequence homology with that of
C. thermocellum
and appears at the same position in the scaffoldin. (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain. Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins. The modular organization of CipV is reminiscent of that of the CipA scaffoldin from
C. thermocellum
as opposed to the known scaffoldins from the mesophilic clostridia. The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.
Influence of
S
-Adenosylmethionine Pool Size on Spontaneous Mutation, Dam Methylation, and Cell Growth of
Escherichia coli
Authors: Posnick Lauren M. , Samson Leona D.
Published: December 2019

Escherichia coli
strains that are deficient in the Ada and Ogt DNA repair methyltransferases display an elevated spontaneous G:C-to-A:T transition mutation rate, and this increase has been attributed to mutagenic
O
6
-alkylguanine lesions being formed via the alkylation of DNA by endogenous metabolites. Here we test the frequently cited hypothesis that
S
-adenosylmethionine (SAM) can act as a weak alkylating agent in vivo and that it contributes to endogenous DNA alkylation. By regulating the expression of the rat liver SAM synthetase and the bacteriophage T3 SAM hydrolase proteins in
E. coli
, a 100-fold range of SAM levels could be achieved. However, neither increasing nor decreasing SAM levels significantly affected spontaneous mutation rates, leading us to conclude that SAM is not a major contributor to the endogenous formation of
O
6
-methylguanine lesions in
E. coli
.
Identification and Disruption of
lisRK
, a Genetic Locus Encoding a Two-Component Signal Transduction System Involved in Stress Tolerance and Virulence in
Listeria monocytogenes
Authors: Cotter Paul D. , Emerson Nathan , Gahan Cormac G. M. , Hill Colin
Published: December 2019

lisRK
encodes a two-component regulatory system in the food pathogen
Listeria monocytogenes
LO28. Following identification of the operon in an acid-tolerant Tn
917
mutant, a deletion in the histidine kinase component was shown to result in a growth phase variation in acid tolerance, an ability to grow in high ethanol concentrations, and a significant reduction in virulence.
Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of
Streptococcus salivarius
Authors: Plamondon Pascale , Brochu Denis , Thomas Suzanne , Fradette Julie , Gauthier Lucie , Vaillancourt Katy , Buckley Nicole , Frenette Michel , Vadeboncoeur Christian
Published: December 2019

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His
15
) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser
46
) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from
Streptococcus salivarius
ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in
ptsH
(the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His
15
by EI nor the phosphorylation at Ser
46
by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.
IS
1397
Is Active for Transposition into the Chromosome of
Escherichia coli
K-12 and Inserts Specifically into Palindromic Units of Bacterial Interspersed Mosaic Elements
Authors: Clément Jean-Marie , Wilde Caroline , Bachellier Sophie , Lambert Patricia , Hofnung Maurice
Published: December 2019

We demonstrate that IS
1397
, a putative mobile genetic element discovered in natural isolates of
Escherichia coli
, is active for transposition into the chromosome of
E. coli
K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to
E. coli
and
Salmonella
. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.
Molecular Cloning and Characterization of the
Helicobacter pylori fliD
Gene, an Essential Factor in Flagellar Structure and Motility
Authors: Seong Kim Jang , Hoon Chang Ji , Il Chung Soo , Sun Yum Jung
Published: December 2019

Helicobacter pylori
colonizes the human stomach and can cause gastroduodenal disease. Flagellar motility is regarded as a major factor in the colonizing ability of
H. pylori
. The functional roles of flagellar structural proteins other than FlaA, FlaB, and FlgE are not well understood. The
fliD
operon of
H. pylori
consists of
flaG
,
fliD
, and
fliS
genes, in the order stated, under the control of a ς
28
-dependent promoter. In an effort to elucidate the function of the FliD protein, a hook-associated protein 2 homologue, in flagellar morphogenesis and motility, the
fliD
gene (2,058 bp) was cloned and isogenic mutants were constructed by disruption of the
fliD
gene with a kanamycin resistance cassette and electroporation-mediated allelic-exchange mutagenesis. In the
fliD
mutant, morphologically abnormal flagellar appendages in which very little filament elongation was apparent were observed. The
fliD
mutant strain was completely nonmotile, indicating that these abnormal flagella were functionally defective. Furthermore, the isogenic
fliD
mutant of
H. pylori
SS1, a mouse-adapted strain, was not able to colonize the gastric mucosae of host mice. These results suggest that
H. pylori
FliD is an essential element in the assembly of the functional flagella that are required for colonization of the gastric mucosa.
TPW22, a Lactococcal Temperate Phage with a Site-Specific Integrase Closely Related to
Streptococcus thermophilus
Phage Integrases
Authors: Petersen Anne , Josephsen Jytte , Johnsen Mads G.
Published: December 2019

The temperate phage TPW22, induced from
Lactococcus lactis
subsp.
cremoris
W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on
L. lactis
subsp.
cremoris
3107, which could also be lysogenized by site-specific integration. The attachment site (
attP
), 5′-TAAGGCGACGGTCG-3′, of phage TPW22 was present on a 7.5-kb
Eco
RI fragment, a 3.4-kb
Eco
RI-
Hin
dIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (
int
). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing
attP
and
int
was constructed, and integration in
L. lactis
subsp.
cremoris
MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages φLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the φLC3 lactococcal integrase with known
Streptococcus thermophilus
integrases.
Functional Regions of the
Bacillus subtilis
Spore Coat Morphogenetic Protein CotE
Authors: Bauer Tamara , Little Shawn , Stöver Axel G. , Driks Adam
Published: December 2019

The
Bacillus subtilis
spore is encased in a resilient, multilayered proteinaceous shell, called the coat, that protects it from the environment. A 181-amino-acid coat protein called CotE assembles into the coat early in spore formation and plays a morphogenetic role in the assembly of the coat’s outer layer. We have used a series of mutant alleles of
cotE
to identify regions involved in outer coat protein assembly. We found that the insertion of a 10-amino-acid epitope, between amino acids 178 and 179 of CotE, reduced or prevented the assembly of several spore coat proteins, including, most likely, CotG and CotB. The removal of 9 or 23 of the C-terminal-most amino acids resulted in an unusually thin outer coat from which a larger set of spore proteins was missing. In contrast, the removal of 37 amino acids from the C terminus, as well as other alterations between amino acids 4 and 160, resulted in the absence of a detectable outer coat but did not prevent localization of CotE to the forespore. These results indicate that changes in the C-terminal 23 amino acids of CotE and in the remainder of the protein have different consequences for outer coat protein assembly.
A Mutation in the 3-Phosphoglycerate Kinase Gene Allows Anaerobic Growth of
Bacillus subtilis
in the Absence of ResE Kinase
Authors: Nakano Michiko M. , Zhu Yi , Haga Koki , Yoshikawa Hirofumi , Sonenshein Abraham L. , Zuber Peter
Published: December 2019

The
Bacillus subtilis
ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to
pgk
, the gene encoding 3-phosphoglycerate kinase, failed to suppress a
resD
mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The
pgk-1
mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1,3-diphosphoglycerate, is responsible for the observed effect of the
pgk-1
mutation on anaerobiosis of
resE
mutant cells.
Isolation, Cloning, and Expression of an Acid Phosphatase Containing Phosphotyrosyl Phosphatase Activity from
Prevotella intermedia
Authors: Chen Xiaochi , Ansai Toshihiro , Awano Shuji , Iida Toshiya , Barik Sailen , Takehara Tadamichi
Published: December 2019

A novel acid phosphatase containing phosphotyrosyl phosphatase (PTPase) activity, designated PiACP, from
Prevotella intermedia
ATCC 25611, an anaerobe implicated in progressive periodontal disease, has been purified and characterized. PiACP, a monomer with an apparent molecular mass of 30 kDa, did not require divalent metal cations for activity and was sensitive to orthovanadate but highly resistant to okadaic acid. The enzyme exhibited substantial activity against tyrosine phosphate-containing peptides derived from the epidermal growth factor receptor. On the basis of N-terminal and internal amino acid sequences of purified PiACP, the gene coding for PiACP was isolated and sequenced. The PiACP gene consisted of 792 bp and coded for a basic protein with an
M
r
of 29,164. The deduced amino acid sequence exhibited striking similarity (25 to 64%) to those of members of class A bacterial acid phosphatases, including PhoC of
Morganella morganii
, and involved a conserved phosphatase sequence motif that is shared among several lipid phosphatases and the mammalian glucose-6-phosphatases. The highly conservative motif HCXAGXXR in the active domain of PTPase was not found in PiACP. Mutagenesis of recombinant PiACP showed that His-170 and His-209 were essential for activity. Thus, the class A bacterial acid phosphatases including PiACP may function as atypical PTPases, the biological functions of which remain to be determined.
Roles of Three Histidine Kinase Genes in Hyphal Development and Virulence of the Pathogenic Fungus
Candida albicans
Authors: Yamada-Okabe Toshiko , Mio Toshiyuki , Ono Naomi , Kashima Yuji , Matsui Mitsuaki , Arisawa Mikio , Yamada-Okabe Hisafumi
Published: December 2019

The pathogenic fungus
Candida albicans
harbors three histidine kinase genes called
CaSLN1
,
CaNIK1
, and
CaHK1
. The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of
C. albicans
in a mouse systemic candidiasis model. The effects of the disruption on hyphal formation and virulence were most severe in the
cahk1
Δ null mutants. Although the double disruption of
CaSLN1
and
CaNIK1
was impossible, further deletion of
CaSLN1
or
CaNIK1
in the
cahk1
Δ null mutants partially restored the serum-induced hypha-forming ability and virulence. When incubated with radiolabelled ATP, the recombinant CaSln1 and CaNik1 proteins, which contained their own kinase and response regulator domains, were autophosphorylated, whereas CaHk1p was not. These results imply that in
C. albicans
,
CaSLN1
and
CaNIK1
function upstream of
CaHK1
but are in distinct signal transmission pathways.
A Novel Nitrate/Nitrite Permease in the Marine Cyanobacterium
Synechococcus
sp. Strain PCC 7002
Authors: Sakamoto Toshio , Inoue-Sakamoto Kaori , Bryant Donald A.
Published: December 2019

The
nrtP
and
narB
genes, encoding nitrate/nitrite permease and nitrate reductase, respectively, were isolated from the marine cyanobacterium
Synechococcus
sp. strain PCC 7002 and characterized. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters that previously have been described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms. An
nrtP
mutant strain consumes nitrate at a 4.5-fold-lower rate than the wild type, and this mutant grew exponentially on a medium containing 12 mM nitrate at a rate approximately 2-fold lower than that of the wild type. The
nrtP
mutant cells could not consume nitrite as rapidly as the wild type at pH 10, suggesting that NrtP also functions in nitrite uptake. A
narB
mutant was unable to grow on a medium containing nitrate as a nitrogen source, although this mutant could grow on media containing urea or nitrite with rates similar to those of the wild type. Exogenously added nitrite enhanced the in vivo activity of nitrite reductase in the
narB
mutant; this suggests that nitrite acts as a positive effector of nitrite reductase. Transcripts of the
nrtP
and
narB
genes were detected in cells grown on nitrate but were not detected in cells grown on urea or ammonia. Transcription of the
nrtP
and
narB
genes is probably controlled by the NtcA transcription factor for global nitrogen control. The discovery of a nitrate/nitrite permease in
Synechococcus
sp. strain PCC 7002 suggests that significant differences in nutrient transporters may occur in marine and freshwater cyanobacteria.
Regulation of
adhE
(Encoding Ethanol Oxidoreductase) by the Fis Protein in
Escherichia coli
Authors: Membrillo-Hernández Jorge , Kwon Ohsuk , De Wulf Peter , Finkel Steven E. , Lin E. C. C.
Published: December 2019

The
adhE
gene of
Escherichia coli
encodes a multifunctional ethanol oxidoreductase whose expression is 10-fold higher under anaerobic than aerobic conditions. Transcription of the gene is under the negative control of the Cra (catabolite repressor-activator) protein, whereas translation of the
adhE
mRNA requires processing by RNase III. In this report, we show that the expression of
adhE
also depends on the Fis (factor for inversion stimulation) protein. A strain bearing a
fis
::
kan
null allele failed to grow anaerobically on glucose solely because of inadequate
adhE
transcription. However,
fis
expression itself is not under redox control. Sequence inspection of the
adhE
promoter revealed three potential Fis binding sites. Electrophoretic mobility shift analysis, using purified Fis protein and
adhE
promoter DNA, showed three different complexes.
Genetic Analysis of a Chromosomal Region Containing Genes Required for Assimilation of Allantoin Nitrogen and Linked Glyoxylate Metabolism in
Escherichia coli
Authors: Cusa Eva , Obradors Nuria , Baldomà Laura , Badía Josefa , Aguilar Juan
Published: December 2019

Growth experiments with
Escherichia coli
have shown that this organism is able to use allantoin as a sole nitrogen source but not as a sole carbon source. Nitrogen assimilation from this compound was possible only under anaerobic conditions, in which all the enzyme activities involved in allantoin metabolism were detected. Of the nine genes encoding proteins required for allantoin degradation, only the one encoding glyoxylate carboligase (
gcl
), the first enzyme of the pathway leading to glycerate, had been identified and mapped at centisome 12 on the chromosome map. Phenotypic complementation of mutations in the other two genes of the glycerate pathway, encoding tartronic semialdehyde reductase (
glxR
) and glycerate kinase (
glxK
), allowed us to clone and map them closely linked to
gcl
. Complete sequencing of a 15.8-kb fragment encompassing these genes defined a regulon with 12 open reading frames (ORFs). Due to the high similarity of the products of two of these ORFs with yeast allantoinase and yeast allantoate amidohydrolase, a systematic analysis of the gene cluster was undertaken to identify genes involved in allantoin utilization. A BLASTP search predicted four of the genes that we sequenced to encode allantoinase (
allB
), allantoate amidohydrolase (
allC
), ureidoglycolate hydrolase (
allA
), and ureidoglycolate dehydrogenase (
allD
). The products of these genes were overexpressed and shown to have the predicted corresponding enzyme activities. Transcriptional fusions to
lacZ
permitted the identification of three functional promoters corresponding to three transcriptional units for the structural genes and another promoter for the regulatory gene
allR
. Deletion of this regulatory gene led to constitutive expression of the regulon, indicating a negatively acting function.
Vir Proteins Stabilize VirB5 and Mediate Its Association with the T Pilus of
Agrobacterium tumefaciens
Authors: Schmidt-Eisenlohr Heike , Domke Natalie , Angerer Christina , Wanner Gerhard , Zambryski Patricia C. , Baron Christian
Published: December 2019

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as putative components of the T pilus from
Agrobacterium tumefaciens
, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C. I. Kado, J. Bacteriol. 180:2711–2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of VirB2 proved to be inherent features of these proteins, independent of virulence gene induction. In contrast, cellular levels of VirB5 were strongly reduced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus were analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulence gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifugation. VirB5 may therefore be directly involved in pilus assembly, possibly as minor component. In contrast, secreted VirB1* showed no association with the T pilus. In-frame deletions in genes
virB1
,
virB2
,
virB5
, and
virB6
blocked the formation of virulence gene-dependent extracellular high-molecular-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.
Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism
Authors: Nemecek-Marshall Michele , Wojciechowski Cheryl , Wagner William P. , Fall Ray
Published: December 2019

There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the
Vibrionaceae
. Sixteen
Vibrio
isolates, two
Listonella
species, and two
Photobacterium angustum
isolates produced acetone in the presence of
l
-leucine.
Shewanella
isolates produced much less acetone. Growth of
Vibrio splendidus
and
P. angustum
in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium.
l
-Leucine, but not
d
-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of
l
-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only α-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d
7
)-
l
-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads.
Differential and Independent Roles of a ς
32
Homolog (RpoH) and an HrcA Repressor in the Heat Shock Response of
Agrobacterium tumefaciens
Authors: Nakahigashi Kenji , Ron Eliora Z. , Yanagi Hideki , Yura Takashi
Published: December 2019

The heat shock response in alpha proteobacteria is unique in that a combination of two regulators is involved: a positive regulator, RpoH (ς
32
homolog), found in the alpha, beta, and gamma proteobacteria, and a negative regulator, HrcA, widely distributed in eubacteria but not in the gamma proteobacteria. To assess the differential roles of the two regulators in these bacteria, we cloned the
hrcA-grpE
operon of
Agrobacterium tumefaciens
, analyzed its transcription, and constructed deletion mutants lacking RpoH and/or HrcA. The
ΔrpoH
mutant and
ΔrpoH ΔhrcA
double mutant were unable to grow above 30°C. Whereas the synthesis of heat shock proteins (e.g., DnaK, GroEL, and ClpB) was transiently induced upon temperature upshift from 25 to 37°C in the wild type, such induction was not observed in the
ΔrpoH
mutant, except that GroEL synthesis was still partially induced. By contrast, the
ΔhrcA
mutant grew normally and exhibited essentially normal heat induction except for a higher level of GroEL expression, especially before heat shock. The
ΔrpoH ΔhrcA
double mutant showed the combined phenotypes of each of the single mutants. The amounts of
dnaK
and
groE
transcripts before and after heat shock, as determined by primer extension, were consistent with those of the proteins synthesized. The cellular level of RpoH but not HrcA increased significantly upon heat shock. We conclude that RpoH plays a major and global role in the induction of most heat shock proteins, whereas HrcA plays a restricted role in repressing
groE
expression under nonstress conditions.
Gradual Alterations in Cell Wall Structure and Metabolism in Vancomycin-Resistant Mutants of
Staphylococcus aureus
Authors: Sieradzki Krzysztof , Tomasz Alexander
Published: December 2019

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant
Staphylococcus aureus
strain COL (MIC of methicillin, 800 μg/ml; MIC of vancomycin, 1.5 μg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to β-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.
Regulation of Expression of the
adhE
Gene, Encoding Ethanol Oxidoreductase in
Escherichia coli
: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS
Authors: Membrillo-Hernández Jorge , Lin E. C. C.
Published: December 2019

The
adhE
gene of
Escherichia coli
, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of
adhE
is dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the
lacZ
gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activates
adhE
transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.
RNase E Enzymes from
Rhodobacter capsulatus
and
Escherichia coli
Differ in Context- and Sequence-Dependent In Vivo Cleavage within the Polycistronic
puf
mRNA
Authors: Heck Claudia , Evguenieva-Hackenberg Elena , Balzer Angelika , Klug Gabriele
Published: December 2019

The 5′
pufQ
mRNA segment and the
pufLMX
mRNA segment of
Rhodobacter capsulatus
exhibit different stabilities. Degradation of both mRNA segments is initiated by RNase E-mediated endonucleolytic cleavage. While
Rhodobacter
RNase E does not discriminate between the different sequences present around the cleavage sites within
pufQ
and
pufL
,
Escherichia coli
RNase E shows preference for the sequence harboring more A and U residues.
Archaeal Nucleosome Positioning by CTG Repeats
Authors: Sandman Kathleen , Reeve John N.
Published: December 2019

DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)
6
and (CTG)
8
repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)
4
-, (CTG)
5
-, or (CTG)
3
AA(CTG)
3
-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.
Membrane-Mimicking Entities Induce Structuring of the Two-Peptide Bacteriocins Plantaricin E/F and Plantaricin J/K
Authors: Hauge Håvard Hildeng , Mantzilas Dimitris , Eijsink Vincent G. H. , Nissen-Meyer Jon
Published: December 2019

Lactobacillus plantarum
C11 produces plantaricin E/F (PlnE/F) and plantaricin J/K (PlnJ/K), two bacteriocins whose activity depends on the complementary action of two peptides (PlnE and PlnF; PlnJ and PlnK). Three of the individual Pln peptides possess some antimicrobial activity, but the highest bacteriocin activity is obtained by combining complementary peptides in about a one-to-one ratio. Circular dichroism was used to study the structure of the Pln peptides under various conditions. All four peptides were unstructured under aqueous conditions but adopted a partly alpha-helical structure in the presence of trifluoroethanol, micelles of dodecylphosphocholine, and negatively charged dioleoylphosphoglycerol (DOPG) liposomes. Far less structure was induced by zwitterionic dioleoylglycerophosphocholine liposomes, indicating that a net negative charge on the phospholipid bilayer is important for a structure-inducing interaction between (positively charged) Pln peptides and a membrane. The structuring of complementary peptides was considerably enhanced when both (PlnE and PlnF or PlnJ and PlnK) were added simultaneously to DOPG liposomes. Such additional structuring was not observed in experiments with trifluoroethanol or dodecylphosphocholine, indicating that the apparent structure-inducing interaction between complementary Pln peptides requires the presence of a phospholipid bilayer. The amino acid sequences of the Pln peptides are such that the alpha-helical structures adopted upon interaction with the membrane and each other are amphiphilic in nature, thus enabling membrane interactions.
Assembly of the K40 Antigen in
Escherichia coli
: Identification of a Novel Enzyme Responsible for Addition of
l
-Serine Residues to the Glycan Backbone and Its Requirement for K40 Polymerization
Authors: Amor Paul A. , Yethon Jeremy A. , Monteiro Mario A. , Whitfield Chris
Published: December 2019

Escherichia coli
O8:K40 coexpresses two distinct lipopolysaccharide (LPS) structures on its surface. The O8 polysaccharide is a mannose homopolymer with a trisaccharide repeat unit and is synthesized by an ABC-2 transport-dependent pathway. The K40
LPS
backbone structure is composed of a trisaccharide repeating unit of
N
-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) and has an uncommon substitution, an
l
-serine moiety attached to glucuronic acid. The gene cluster responsible for synthesis of the K40 polysaccharide has previously been cloned and sequenced and was found to contain six open reading frames (ORFs) (P. A. Amor and C. Whitfield, Mol. Microbiol. 26:145–161, 1997). Here, we demonstrate that insertional inactivation of
orf1
results in the accumulation of a semirough (SR)-K40
LPS
form which retains reactivity with specific polyclonal serum in Western immunoblots. Structural and compositional analysis of the SR-K40
LPS
reveals that it comprises a single K40 repeat unit attached to lipid A core. The lack of polymerization of the K40 polysaccharide indicates that
orf1
encodes the K40 polymerase (Wzy) and that assembly of the K40 polysaccharide occurs via a Wzy-dependent pathway (in contrast to that of the O8 polysaccharide). Inactivation of
orf3
also results in the accumulation of an SR-LPS form which fails to react with specific polyclonal K40 serum in Western immunoblots. Methylation linkage analysis and fast atom bombardment-mass spectrometry of this SR-LPS reveals that the biological repeat unit of the K40 polysaccharide is GlcNAc-GlcA-GlcNAc. Additionally, this structure lacks the
l
-serine substitution of GlcA. These results show that (i)
orf3
encodes the enzyme responsible for the addition of the
l
-serine residue to the K40 backbone and (ii) substitution of individual K40 repeats with
l
-serine is essential for their recognition and polymerization into the K40 polysaccharide by Wzy.
A Four-Dimensional View of Assembly of a Morphogenetic Protein during Sporulation in
Bacillus subtilis
Authors: Price Kirsten D. , Losick Richard
Published: December 2019

We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium
Bacillus subtilis
. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium
Clostridium acetobutylicum
, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like
spoIVA
, are under the control of the mother cell transcription factor ς
E
. One such gene was
spoVM
, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.
Coordinate Intracellular Expression of
Salmonella
Genes Induced during Infection
Authors: Heithoff Douglas M. , Conner Christopher P. , Hentschel Ute , Govantes Fernando , Hanna Philip C. , Mahan Michael J.
Published: December 2019

Salmonella typhimurium
in vivo-induced (
ivi
) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg
2+
, Fe
2+
, and PhoPQ. All of the seven
ivi
fusions that are induced by both low pH and low Mg
2+
(e.g.,
iviVI-A
) are activated by the PhoPQ regulatory system. Iron-responsive
ivi
fusions include those induced under iron limitation (e.g.,
entF
) as well as one induced by iron excess but only in the absence of PhoP (
pdu
). Intracellular expression studies showed that each of the pH- and Mg
2+
-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407. Each
ivi
fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed
ivi
genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types. Investigation of
ivi
expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments.
Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in
Salmonella typhimurium
: Incorporation of Stable Isotope-Labeled Glycine and Formate
Authors: Enos-Berlage Jodi L. , Downs Diana M.
Published: December 2019

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in
Salmonella typhimurium
. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-
13
C]glycine and [
13
C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a
purF
mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.
Poly-3-Hydroxybutyrate Degradation in
Rhizobium (Sinorhizobium) meliloti
: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase
Authors: Aneja P. , Charles T. C.
Published: December 2019

We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (
bdhA
) from
Rhizobium (Sinorhizobium) meliloti
. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The
R. meliloti
Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily.
bdhA
is the first gene transcribed in an operon that also includes
xdhA
, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς
54
consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a
rpoN
mutant of
R. meliloti
was able to utilize 3-hydroxybutyrate as a sole carbon source. The
R. meliloti bdhA
gene is able to confer upon
Escherichia coli
the ability to utilize 3-hydroxybutyrate as a sole carbon source. An
R. meliloti bdhA
mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying a
lacZ
transcriptional fusion to
bdhA
demonstrated that gene expression is growth phase associated.
Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium
Authors: Adams David G. , Ashworth Denise , Nelmes Brenda
Published: December 2019

The cell walls of a number of filamentous, gliding cyanobacteria of the genusOscillatoriawere examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.
Diversity of
radA
Genes from Cultured and Uncultured
Archaea
: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker
Authors: Sandler Steven J. , Hugenholtz Philip , Schleper Christa , DeLong Edward F. , Pace Norman R. , Clark Alvin J.
Published: December 2019

Archaea
-specific
radA
primers were used with PCR to amplify fragments of
radA
genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15
radA
genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of
Crenarcheota
. The two most deeply branching archaeal
radA
genes found had some amino acid deletion and insertion patterns characteristic of bacterial
recA
genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.