bluuebunny/crossref_metadata_2025 huggingface dataset:
A
Zymomonas mobilis
Mutant with Delayed Growth on High Glucose Concentrations
Authors: Douka Eugenia , Koukkou Anna Irini , Vartholomatos Georgios , Frillingos Stathis , Papamichael Emmanuel M. , Drainas Constantin
Published: December 2019

Exponentially growing cells of
Zymomonas mobilis
normally exhibit a lag period of up to 3 h when transferred from 0.11 M (2%) to 0.55 M (10%) glucose liquid medium. A mutant of
Z. mobilis
(CU1Rif2), fortuitously isolated, showed more than a 20-h lag period when grown under the same conditions, whereas on 0.55 M glucose solid medium, it failed to grow. The growth of CU1Rif2 on elevated concentrations of other fermentable (0.55 M sucrose or fructose) or nonfermentable (0.11 M glucose plus 0.44 M maltose or xylose) sugars appeared to be normal. Surprisingly, CU1Rif2 cells grew without any delay on 0.55 M glucose on which wild-type cells had been incubated for 3 h and removed at the beginning of their exponential phase. This apparent preconditioning was not observed with medium obtained from wild-type cells grown on 0.11 M glucose and supplemented to 0.55 M after removal of the wild-type cells. Undelayed growth of CU1Rif2 on 0.55 M glucose previously conditioned by the wild type was impaired by heating or protease treatment. It is suggested that in
Z. mobilis
, a diffusible proteinaceous heat-labile factor, transitionally not present in 0.55 M glucose CU1Rif2 cultures, triggers growth on 0.55 M glucose. Biochemical analysis of glucose uptake and glycolytic enzymes implied that glucose assimilation was not directly involved in the phenomenon. By use of a wild-type
Z. mobilis
genomic library, a 4.5-kb DNA fragment which complemented in low copy number the glucose-defective phenotype as well as glucokinase and glucose uptake of CU1Rif2 was isolated. This fragment carries a gene cluster consisting of four putative coding regions, encoding 167, 167, 145, and 220 amino acids with typical
Z. mobilis
codon usage, −35 and −10 promoter elements, and individual Shine-Dalgarno consensus sites. However, strong homologies were not detected in a BLAST2 (EMBL-Heidelberg) computer search with known protein sequences.
The
Saccharomyces cerevisiae
Weak-Acid-Inducible ABC Transporter Pdr12 Transports Fluorescein and Preservative Anions from the Cytosol by an Energy-Dependent Mechanism
Authors: Holyoak Caroline D. , Bracey Danielle , Piper Peter W. , Kuchler Karl , Coote Peter J.
Published: December 2019

Growth of
Saccharomyces cerevisiae
in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257–4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C
1
to C
7
. Exposure to acids with aliphatic chain lengths greater than C
7
resulted in no observable sensitivity of
Δpdr12
mutant cells compared to the parent. Parent and
Δpdr12
mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the
Δpdr12
mutant were unable to efflux any of the dye. When both parent and
Δpdr12
mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.
Obg, an Essential GTP Binding Protein of
Bacillus subtilis
, Is Necessary for Stress Activation of Transcription Factor ς
B
Authors: Scott Janelle M. , Haldenwang W. G.
Published: December 2019

ς
B
, the general stress response ς factor of
Bacillus subtilis
, is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Stress activates ς
B
by means of a collection of regulatory kinases and phosphatases (the Rsb proteins), which catalyze the release of ς
B
from an anti-ς factor inhibitor. By using the yeast dihybrid selection system to identify
B. subtilis
proteins that could interact with Rsb proteins and act as mediators of stress signaling, we isolated the GTP binding protein, Obg, as an interactor with several of these regulators (RsbT, RsbW, and RsbX).
B. subtilis
depleted of Obg no longer activated ς
B
in response to environmental stress, but it retained the ability to activate ς
B
by the ATP responsive pathway. Stress pathway components activated ς
B
in the absence of Obg if the pathway’s most upstream effector (RsbT) was synthesized in excess to the inhibitor (RsbS) from which it is normally released after stress. Thus, the Rsb proteins can function in the absence of Obg but fail to be triggered by stress. The data demonstrate that Obg, or a process under its control, is necessary to induce the stress-dependent activation of ς
B
and suggest that Obg may directly communicate with one or more ς
B
regulators.
Complementation of Conjugation Functions of
Streptomyces lividans
Plasmid pIJ101 by the Related
Streptomyces
Plasmid pSB24.2
Authors: Pettis Gregg S. , Prakash Shubha
Published: December 2019

A database search revealed extensive sequence similarity between
Streptomyces lividans
plasmid pIJ101 and
Streptomyces
plasmid pSB24.2, which is a deletion derivative of
Streptomyces cyanogenus
plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the
cis
-acting locus for transfer (
clt
) and the transfer-associated
korB
gene, were shown to be capable of complementing the pIJ101
clt
and
korB
functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.
Involvement of the RNA Polymerase α-Subunit C-Terminal Domain in LuxR-Dependent Activation of the
Vibrio fischeri
Luminescence Genes
Authors: Stevens Ann M. , Fujita Nobuyuki , Ishihama Akira , Greenberg E. P.
Published: December 2019

LuxR is a ς
70
RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the
Vibrio fischeri lux
operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the α-subunit C-terminal domain (αCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRΔN) was used to study the activation of transcription from the
luxI
promoter in vitro. Initiation of
lux
operon transcription was observed in the presence of LuxRΔN and wild-type RNAP but not in the presence of LuxRΔN and RNAPs with truncated αCTDs. We also studied the in vivo role of the RNAP αCTD in activation of
lux
transcription in
Escherichia coli
. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRΔN. These in vivo studies indicated that both LuxR and LuxRΔN require the RNAP αCTD for activity. The results of DNase I protection studies showed that LuxRΔN-RNAP complexes can bind and protect the
luxI
promoter, but with less efficacy when the αCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the
lux
operon involves the RNAP αCTD and suggest that αCTD-LuxR interactions may play a role in recruitment of RNAP to the
luxI
promoter.
Serotype 1a O-Antigen Modification: Molecular Characterization of the Genes Involved and Their Novel Organization in the
Shigella flexneri
Chromosome
Authors: Adhikari Pradip , Allison Gwen , Whittle Belinda , Verma Naresh K.
Published: December 2019

The factors responsible for serotype 1a O-antigen modification in
Shigella flexneri
were localized to a 5.8-kb chromosomal
Hin
dIII fragment of serotype 1a strain Y53. The entire 5.8-kb fragment and regions up- and downstream of it (10.6-kb total) were sequenced. A putative three-gene operon, which showed homology with other serotype conversion genes, was identified and shown to confer serotype 1a O-antigen modification. The serotype conversion genes were flanked on either side by phage DNA. Multiple insertion sequence (IS) elements were located within and upstream of the phage DNA in a composite transposon-like structure. Host DNA homologous to the
dsdC
and the
thrW proA
genes was located upstream of the IS elements and downstream of the phage DNA, respectively. The sequence analysis indicates that the organization of the 10.6-kb region of the Y53 chromosome is unique and suggests that the serotype conversion genes were originally brought into the host by a bacteriophage. Several features of this region are also characteristic of pathogenicity islands.
RpoS Synthesis Is Growth Rate Regulated in Salmonella typhimurium, but Its Turnover Is Not Dependent on Acetyl Phosphate Synthesis or PTS Function
Authors: Cunning Christofer , Elliott Thomas
Published: December 2019

The RpoS sigma factor of enteric bacteria is either required for or augments the expression of a number of genes that are induced during nutrient limitation, growth into stationary phase, or in response to stresses, including high osmolarity. RpoS is regulated at multiple levels, including posttranscriptional control of its synthesis, protein turnover, and mechanisms that affect its activity directly. Here, the control of RpoS stability was investigated inSalmonella typhimuriumby the isolation of a number of mutants specifically defective in RpoS turnover. These included 20 mutants defective inmviA, the ortholog ofEscherichia coli rssB/sprE, and 13 mutants defective in eitherclpPorclpXwhich encode the protease active on RpoS. Anhnsmutant was also defective in RpoS turnover, thus confirming thatS. typhimuriumandE. colihave identical genetic requirements for this process. Some current models predict the existence of a kinase to phosphorylate the response regulator MviA, but no mutants affecting a kinase were recovered. AnmviAmutant carrying the D58N substitution altering the predicted phosphorylation site is substantially defective, suggesting that phosphorylation of MviA on D58 is important for its function. No evidence was obtained to support models in which acetyl phosphate or the PTS system contributes to MviA phosphorylation. However, we did find a significant (fivefold) elevation of RpoS during exponential growth on acetate as the carbon and energy source. This behavior is due to growth rate-dependent regulation which increases RpoS synthesis at slower growth rates. Growth rate regulation operates at the level of RpoS synthesis and is mainly posttranscriptional but, surprisingly, is independent ofhfqfunction.
Identification of a Gene in Staphylococcus xylosus Encoding a Novel Glucose Uptake Protein
Authors: Fiegler Heike , Bassias Joannis , Jankovic Ivana , Brückner Reinhold
Published: December 2019

By transposon Tn917mutagenesis, two mutants ofStaphylococcus xylosuswere isolated that showed higher levels of β-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designatedglcU, encoding a protein involved in glucose uptake inS. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of β-galactosidase, α-glucosidase, and β-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons intoglcUreduced glucose dehydrogenase activity, suggesting cotranscription ofglcUandgdh. Insertional inactivation of thegdhgene and deletion of theglcUgene without affectinggdhexpression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in theglcUmutant strains. With clonedglcU, anEscherichia colimutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized byglcUin nonphosphorylated form. A gene fromBacillus subtilis,ycxE, that is homologous toglcU, could substitute forglcUin theE. coliglucose growth experiments and restored glucose repression in theS. xylosus glcUmutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.
Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica
Authors: Perkins-Balding Donna , Duval-Valentin Guy , Glasgow Anna C.
Published: December 2019

The gram-negative marine bacteriumPseudoalteromonas atlanticaproduces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production inP. atlantica. Examination of IS492transposition inP. atlanticaby using a PCR-based assay revealed a circular form of IS492that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492insertion sites on theP. atlanticachromosome that we identified by using inverse PCR. IS492circle formation correlated with precise excision of IS492from theP. atlantica epstarget sequence when introduced intoEscherichia colion a plasmid. Deletion analyses of the flanking host sequences at theepsinsertion site for IS492demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492and circle formation inE. coli. Excision of IS492inE. colialso depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492is suggested by the creation of a new strong promoter for expression ofmooVby the joining of the ends of the insertion sequence element at the circle junction.
Sigma Factor Displacement from RNA Polymerase during Bacillus subtilis Sporulation
Authors: Ju Jingliang , Mitchell Theresa , Peters Howard , Haldenwang W. G.
Published: December 2019

AsBacillus subtilisproceeds through sporulation, the principal vegetative cell ς subunit (ςA) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific ς factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with ςAand two mother cell ς factors, ςEand ςK, which successively replace ςAon RNAP. Although the relative abundance of ςAwith respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable ςAwhich cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T0) to 2 to 8% by 3 h into the process (T3). In a strain that failed to synthesize ςE, the first of the mother cell-specific ς factors, approximately 40% of the ςAremained associated with RNAP atT3. The level of ςA-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a ςEvariant (ςECR119) that could bind to RNAP but was unable to direct ςE-dependent transcription. The E-ςE-to-E-ςKchangeover was characterized by both the displacement of ςEfrom RNAP and the disappearance of ςEfrom the cell. Analyses of extracts from wild-type and mutantB. subtilisshowed that the ςKprotein is required for the displacement of ςEfrom RNAP and also confirmed that ςKis needed for the loss of the ςEprotein. The results indicate that the successive appearance of mother cell ς factors, but not necessarily their activities, is an important element in the displacement of preexisting ς factors from RNAP. It suggests that competition for RNAP by consecutive sporulation ς factors may be an important feature of the holoenzyme changeovers that occur during sporulation.
Transcriptional Analysis of the hmw Gene Cluster of Mycoplasma pneumoniae
Authors: Waldo Robert H. , Popham Phillip L. , Romero-Arroyo Cynthia E. , Mothershed Elizabeth A. , Lee Kyungok K. , Krause Duncan C.
Published: December 2019

Mycoplasma pneumoniae adherence to host cells is a multifactorial process that requires the cytadhesin P1 and additional accessory proteins. The hmw gene cluster consists of the genes p30, hmw3, and hmw1, the products of which are known to be essential for cytadherence, therpsD gene, and six open reading frames of unknown function. Putative transcriptional terminators flank this locus, raising the possibility that these genes are expressed as a single transcriptional unit. However, S1 nuclease protection and primer extension experiments identified probable transcriptional start sites upstream of thep32, p21, p50, and rpsDgenes. Each was preceded at the appropriate spacing by the −10-like sequence TTAAAATT, but the −35 regions were not conserved. Analysis of the M. pneumoniae genome sequence indicated that this promoter-like sequence is found upstream of only a limited number of open reading frames, including the genes for P65 and P200, which are structurally related to HMW1 and HMW3. Promoter deletion studies demonstrated that the promoter-like region upstream ofp21 was necessary for the expression of p30 and an hmw3-cat fusion in M. pneumoniae, while deletion of the promoter-like region upstream of p32 had no apparent effect. Analysis by reverse transcription-PCR confirmed transcriptional linkage of all the open reading frames in thehmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts.
Identification of a New Regulator inStreptococcus pneumoniae Linking Quorum Sensing to Competence for Genetic Transformation
Authors: Lee Myeong S. , Morrison Donald A.
Published: December 2019

Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB,cilC, cilD, cilE,cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at −10 and −25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE andcomAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA,cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comXexpression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes
Authors: Kinoshita Hiroshi , Tsuji Tomohiro , Ipposhi Hiroomi , Nihira Takuya , Yamada Yasuhiro
Published: December 2019

BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barBupstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barBtranslational start codon. The BARE located on the barAupstream region (BARE-3) was found 101 to 76 bp upstream of thebarA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barBtranscription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.
Disruption of narG, the Gene Encoding the Catalytic Subunit of Respiratory Nitrate Reductase, Also Affects Nitrite Respiration in Pseudomonas fluorescens YT101
Authors: Ghiglione Jean-François , Philippot Laurent , Normand Philippe , Lensi Robert , Potier Patrick
Published: December 2019

The Pseudomonas fluorescens YT101 genenarG, which encodes the catalytic α subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar− mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N2O respiration was not affected, anaerobic growth with NO2 as the sole electron acceptor was delayed for all of the Nar− mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.
Functional Interaction between PomA and PomB, the Na+-Driven Flagellar Motor Components of Vibrio alginolyticus
Authors: Yorimitsu Tomohiro , Sato Ken , Asai Yukako , Kawagishi Ikuro , Homma Michio
Published: December 2019

Four proteins, PomA, PomB, MotX, and MotY, appear to be involved in force generation of the sodium-driven polar flagella of Vibrio alginolyticus. Among these, PomA and PomB seem to be associated and to form a sodium channel. By using antipeptide antibodies against PomA or PomB, we carried out immunoprecipitation to verify whether these proteins form a complex and examined the in vivo stabilities of PomA and PomB. As a result, we could demonstrate that PomA and PomB functionally interact with each other.
DsbA Is Required for Stable Expression of Outer Membrane Protein YscC and for Efficient Yop Secretion inYersinia pestis
Authors: Jackson Michael W. , Plano Gregory V.
Published: December 2019

The role of the periplasmic disulfide oxidoreductase DsbA in Yop secretion was investigated in Yersinia pestis. A Y. pestis dsbA mutant secreted reduced amounts of the V antigen and Yops and expressed reduced amounts of the full-sized YscC protein. Site-directed mutagenesis of the four cysteine residues present in the YscC protein resulted in defects similar to those found in thedsbA mutant. These results suggest that YscC contains at least one disulfide bond that is essential for the function of this protein in Yop secretion.
Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of
Streptococcus agalactiae
Type Ia
Authors: Yamamoto Shin , Miyake Katsuhide , Koike Yoichi , Watanabe Masaki , Machida Yuichi , Ohta Michio , Iijima Shinji
Published: December 2019

The type-specific capsular polysaccharide (CP) of a group B streptococcus,
Streptococcus agalactiae
type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[α-
d
-Neu
p
NAc-(2→3)-β-
d
-Gal
p
-(1→4)-β-
d
-Glc
p
NAc-(1→3)]-β-
d
-Gal
p
-(1→4)-β-
d
-Glc
p
-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (
cps
) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated
cpsIaA
to
cpsIaL
,
neu
(neuraminic acid synthesis gene)
A
to
D
,
orf1
and
ung
(uracil DNA glycosylase). The
cps
gene products of
S. agalactiae
type Ia were homologous to proteins involved in CP synthesis of
S. agalactiae
type III and
S. pneumoniae
serotype 14. Unlike the
cps
gene cluster of
S. pneumoniae
serotype 14, transcription of this operon may start from
cpsIaA
,
cpsIaE
, and
orf1
because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3′ end of this operon (downstream of
orf1
and
ung
). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in
Escherichia coli
JM109 harboring plasmids containing various
S. agalactiae
type Ia
cps
gene fragments. Enzyme assays suggested that the gene products of
cpsIaE
,
cpsIaG
,
cpsIaI
, and
cpsIaJ
are putative glucosyltransferase, β-1,4-galactosyltransferase, β-1,3-
N
-acetylglucosaminyltransferase, and β-1,4-galactosyltransferase, respectively.
Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete
Thermomonospora chromogena
and Evidence for Horizontal Transfer of an Entire rRNA Operon
Authors: Yap Wai Ho , Zhang Zhenshui , Wang Yue
Published: December 2019

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete
Thermomonospora chromogena
. The genome of
T. chromogena
contains six rRNA operons (
rrn
), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon
rrnB
exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of
rrnB
is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between
rrnB
operon of
T. chromogena
and
rrnA
operon of another thermophilic actinomycete
Thermobispora bispora
. We propose that
T. chromogena
acquired
rrnB
operon from
T. bispora
or a related organism via horizontal gene transfer.
The Cpx Envelope Stress Response Is Controlled by Amplification and Feedback Inhibition
Authors: Raivio Tracy L. , Popkin Daniel L. , Silhavy Thomas J.
Published: December 2019

In
Escherichia coli
, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the
cpxRA
operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription of
cpxRA
. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.
Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of
Clostridium thermocellum
: Evidence for Gene Duplication and Recombination
Authors: Kataeva Irina , Li Xin-Liang , Chen Huizhong , Choi Sang-Ki , Ljungdahl Lars G.
Published: December 2019

The cellulolytic and hemicellulolytic complex of
Clostridium thermocellum
, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene,
celK
, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of
cbhA
(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate that
celK
and
cbhA
in the genome of
C. thermocellum
have evolved through gene duplication and recombination of domain coding sequences.
celK
without a dockerin domain was expressed in
Escherichia coli
and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzed
p
-nitrophenyl-β-
d
-cellobioside with a

K
m

and a
V
max
of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a

K
i

of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.
Roles of RuvC and RecG in Phage λ Red-Mediated Recombination
Authors: Poteete Anthony R. , Fenton Anita C. , Murphy Kenan C.
Published: December 2019

The recombination properties of
Escherichia coli
strains expressing the
red
genes of bacteriophage λ and lacking
recBCD
function either by mutation or by expression of λ
gam
were examined. The substrates for recombination were nonreplicating λ chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in

ruvC
+
recG
+

,
ruvC recG
+
,

ruvC
+
recG

, and
ruvC recG
hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome’s restriction site marker. In this case, recombinant formation was found to be partially dependent upon
ruvC
function, especially in a
recG
mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the
cat
gene, with flanking
lac
sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher in
recG
than in
recG
+
. These observations favor models in which RecG tends to push invading 3′-ended strands back out of recombination intermediates.
New Sporulation Loci in
Streptomyces coelicolor
A3(2)
Authors: Ryding N. Jamie , Bibb Maureen J. , Molle Virginie , Findlay Kim C. , Chater Keith F. , Buttner Mark J.
Published: December 2019

Sporulation mutants of
Streptomyces coelicolor
appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (
whi
) mutants had been used to define eight sporulation loci,
whiA
,
whiB
,
whiD
,
whiE
,
whiG
,
whiH
,
whiI
, and
whiJ
(K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new
whi
loci, we mutagenized
S. coelicolor
M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115
whi
mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned
whi
genes, self-transmissible SCP2*-derived plasmids carrying
whiA
,
whiB
,
whiG
,
whiH
, or
whiJ
(but not
whiD
,
whiE
, or
whiI
) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31
whi
mutants, an SCP2* library of wild-type
S. coelicolor
chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated
whiK
,
whiL
,
whiM
,
whiN
, and
whiO
. Each of the five loci was located on the ordered cosmid library:
whiL
,
whiM
,
whiN
, and
whiO
occupied positions distinct from previously cloned
whi
genes;
whiK
was located on the same cosmid overlap as
whiD
, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.
Mutations Affecting Motifs of Unknown Function in the Central Domain of Nitrogen Regulatory Protein C
Authors: Li Jieli , Passaglia Luciane , Rombel Irene , Yan Dalai , Kustu Sydney
Published: December 2019

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of ς
54
holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated
ntrC
genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of
glnA
transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrC
WT
). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrC
WT
at low concentrations. Their elevated activation capacities remain to be explained.
First Evidence for the Presence of a Hydrogenase in the Sulfur-Reducing Bacterium
Desulfuromonas acetoxidans
Authors: Brugna Marianne , Nitschke Wolfgang , Toci René , Bruschi Mireille , Giudici-Orticoni Marie-Thérèse
Published: December 2019

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from
Desulfuromonas acetoxidans
. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium
D. acetoxidans
, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of
D. acetoxidans
, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.
Population Genetic and Evolutionary Approaches to Analysis of
Neisseria meningitidis
Isolates Belonging to the ET-5 Complex
Authors: Bygraves J. A. , Urwin R. , Fox A. J. , Gray S. J. , Russell J. E. , Feavers I. M. , Maiden M. C. J.
Published: December 2019

Periodically, new disease-associated variants of the human pathogen
Neisseria meningitidis
arise. These meningococci diversify during spread, and related isolates recovered from different parts of the world have different genetic and antigenic characteristics. An example is the ET-5 complex, members of which were isolated globally from the mid-1970s onwards. Isolates from a hyperendemic outbreak of meningococcal disease in Worcester, England, during the late 1980s were characterized by multilocus sequence typing and sequence determination of antigen genes. These data established that the Worcester outbreak was caused by ET-5 complex meningococci which were not closely related to the ET-5 complex bacteria responsible for a hyperendemic outbreak in the nearby town of Stroud during the years preceding the Worcester outbreak. A comparison with other ET-5 complex meningococci established that there were at least three distinct globally distributed subpopulations within the ET-5 complex, characterized by particular housekeeping and antigen gene alleles. The Worcester isolates belonged to one of these subpopulations, the Stroud isolates belonged to another, and at least one representative of the third subpopulation identified in this work was isolated elsewhere in the United Kingdom. The sequence data demonstrated that ET-5 variants have arisen by multiple complex pathways involving the recombination of antigen and housekeeping genes and de novo mutation of antigen genes. The data further suggest that either the ET-5 complex has been in existence for many years, evolving and spreading relatively slowly until its disease-causing potential was recognized, or it has evolved and spread rapidly since its first identification in the 1970s, with each of the subpopulations attaining a distribution spanning several continents.
Cloning and Molecular Characterization of the Genes for Carbon Monoxide Dehydrogenase and Localization of Molybdopterin, Flavin Adenine Dinucleotide, and Iron-Sulfur Centers in the Enzyme of
Hydrogenophaga pseudoflava
Authors: Kang Beom S. , Kim Young M.
Published: December 2019

Carbon monoxide dehydrogenases (CO-DH) are the enzymes responsible for the oxidation of CO to carbon dioxide in carboxydobacteria and consist of three nonidentical subunits containing molybdopterin flavin adenine dinucleotide (FAD), and two different iron-sulfur clusters (O. Meyer, K. Frunzke, D. Gadkari, S. Jacobitz, I. Hugendieck, and M. Kraut, FEMS Microbiol. Rev. 87:253–260, 1990). The three structural genes of CO-DH in
Hydrogenophaga pseudoflava
were cloned and characterized. The genes were clustered on the chromosome in the transcriptional order
cutM-cutS-cutL
. The cloned
cutM
,
cutS
, and
cutL
genes had open reading frames of 864, 492, and 2,412 nucleotides, coding for proteins with calculated molecular weights of 30,694, 17,752, and 87,224, respectively. The overall identities in the nucleotide sequence of the genes and the amino acid sequence of the subunits with those of other carboxydobacteria were 64.5 to 74.3% and 62.8 to 72.3%, respectively. Primer extension analysis revealed that the transcriptional start site of the genes was the nucleotide G located 47 bp upstream of the
cutM
start codon. The deduced amino acid sequences of the three subunits of CO-DH implied the presence of molybdenum cofactor, FAD, and iron-sulfur centers in CutL, CutM, and CutS, respectively. Fluorometric analysis coupled with denaturing polyacrylamide gel electrophoresis of fractions from hydroxyapatite column chromatography in the presence of 8 M urea of active CO-DH and from gel filtration of spontaneously inactivated enzyme revealed that the large and medium subunits of CO-DH in
H. pseudoflava
bind molybdopterin and FAD cofactors, respectively. Iron-sulfur centers of the enzyme were identified to be present in the small subunit on the basis of the iron content in each subunit eluted from the denaturing polyacrylamide gels.
Genetic Diversity and Mosaicism at the
por
Locus of
Neisseria gonorrhoeae
Authors: Fudyk Trevor C. , Maclean Ian W. , Simonsen J. Neil , Njagi Ephantus N. , Kimani Joshua , Brunham Robert C. , Plummer Francis A.
Published: December 2019

The
por
genes of the predominant serovars of
Neisseria gonorrhoeae
circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of
por
gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However,
por
mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different
por
sequences may be a important strategy by which
N. gonorrhoeae
generates
por
gene diversity within core group populations. Despite extensive sequence variability,
por
expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among
por
alleles may also be finite.
P1 ParB Domain Structure Includes Two Independent Multimerization Domains
Authors: Surtees Jennifer A. , Funnell Barbara E.
Published: December 2019

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in
Escherichia coli
. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.
Identification and Characterization of a Determinant (
eep
) on the
Enterococcus faecalis
Chromosome That Is Involved in Production of the Peptide Sex Pheromone cAD1
Authors: An Florence Y. , Sulavik Mark C. , Clewell Don B.
Published: December 2019

Plasmid-free strains of
Enterococcus faecalis
secrete a peptide sex pheromone, cAD1, which specifically induces a mating response by donors carrying the hemolysin plasmid pAD1 or related elements. A determinant on the
E. faecalis
OG1X chromosome has been found to encode a 46.5-kDa protein that plays an important role in the production of the extracellular cAD1. Wild-type
E. faecalis
OG1X cells harboring a plasmid chimera carrying the determinant exhibited an eightfold enhanced production of cAD1, and plasmid-free cells carrying a mutated chromosomal determinant secreted undetectable or very low amounts of the pheromone. The production of other pheromones such as cPD1, cOB1, and cCF10 was also influenced, although there was no effect on the pheromone cAM373. The determinant, designated
eep
(for enhanced expression of pheromone), did not include the sequence of the pheromone. Its deduced product (Eep) contains apparent membrane-spanning sequences; conceivably it is involved in processing a pheromone precursor structure or in some way regulates expression or secretion.
Transcriptional Control of
Bacillus subtilis hemN
and
hemZ
Authors: Homuth Georg , Rompf Alexandra , Schumann Wolfgang , Jahn Dieter
Published: December 2019

Previous characterization of
Bacillus subtilis hemN
, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second
hemN
-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181–7185, 1997). The corresponding
hemZ
gene was found to be split into the two potential open reading frames
yhaV
and
yhaW
by a sequencing error of the genome sequencing project. The
hemZ
gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a
Salmonella typhimurium hemF hemN
double mutant under aerobic and anaerobic growth conditions. A
B. subtilis hemZ
mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A
hemN hemZ
double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The
hemY
gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of
hemY
mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in
B. subtilis
. A monocistronic
hemZ
mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of
hemZ
and
hemN
demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing
B. subtilis
. The
B. subtilis
redox regulatory systems encoded by
resDE
,
fnr
, and
ywiD
were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via
resDE
, through
fnr
and
ywiD
to
hemN/hemZ
, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only
hemZ
was found to be fivefold induced by the presence of H
2
O
2
, indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.