bluuebunny/crossref_metadata_2025_split huggingface dataset:
Translational Control of Tetracycline Resistance and Conjugation in the
Bacteroides
Conjugative Transposon CTnDOT
Authors: Wang Yanping , Rotman Ella R. , Shoemaker Nadja B. , Salyers Abigail A. 
Published: April 2005

ABSTRACT

The
tetQ-rteA-rteB
operon of the
Bacteroides
conjugative transposon CTnDOT is responsible for tetracycline control of the excision and transfer of CTnDOT. Previous studies revealed that tetracycline control of this operon occurred at the translational level and involved a hairpin structure located within the 130-base leader sequence that lies between the promoter of
tetQ
and the start codon of the gene. This hairpin structure is formed by two sequences, designated Hp1 and Hp8. Hp8 contains the ribosome binding site for
tetQ
. Examination of the leader region sequence revealed three sequences that might encode a leader peptide. One was only 3 amino acids long. The other two were 16 amino acids long. By introducing stop codons into the peptide coding regions, we have now shown that the 3-amino-acid peptide is the one that is essential for tetracycline control. Between Hp1 and Hp8 lies an 85-bp region that contains other possible RNA hairpin structures. Deletion analysis of this intervening DNA segment has now identified a sequence, designated Hp2, which is essential for tetracycline regulation. This sequence could form a short hairpin structure with Hp1. Mutations that made the Hp1-Hp2 structure more stable caused nearly constitutively high expression of the operon. Thus, stalling of ribosomes on the 3-amino-acid leader peptide could favor formation of the Hp1-Hp2 structure and thus preclude formation of the Hp1-Hp8 structure, releasing the ribosome binding site of
tetQ
. Finally, comparison of the CTnDOT
tetQ
leader regions with upstream regions of five
tetQ
genes found in other elements reveals that the sequences are virtually identical, suggesting that translational attenuation is responsible for control of tetracycline resistance in these other cases as well.
Import of the Transfer RNase Colicin D Requires Site-Specific Interaction with the Energy-Transducing Protein TonB
Authors: Mora Liliana , Diaz Nancy , Buckingham Richard H. , de Zamaroczy Miklos 
Published: April 2005

ABSTRACT

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous
tonB
mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both
tonB
mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.
Nucleotide Substitution and Recombination at Orthologous Loci in
Staphylococcus aureus
Authors: Hughes Austin L. , Friedman Robert 
Published: April 2005

ABSTRACT

The pattern of nucleotide substitution was examined at 2,129 orthologous loci among five genomes of
Staphylococcus aureus
, which included two sister pairs of closely related genomes (MW2/MSSA476 and Mu50/N315) and the more distantly related MRSA252. A total of 108 loci were unusual in lacking any synonymous differences among the five genomes; most of these were short genes encoding proteins highly conserved at the amino acid sequence level (including many ribosomal proteins) or unknown predicted genes. In contrast, 45 genes were identified that showed anomalously high divergence at synonymous sites. The latter genes were evidently introduced by homologous recombination from distantly related genomes, and in many cases, the pattern of nucleotide substitution made it possible to reconstruct the most probable recombination event involved. These recombination events introduced genes encoding proteins that differed in amino acid sequence and thus potentially in function. Several of the proteins are known or likely to be involved in pathogenesis (e.g., staphylocoagulase, exotoxin, Ser-Asp fibrinogen-binding bone sialoprotein-binding protein, fibrinogen and keratin-10 binding surface-anchored protein, fibrinogen-binding protein ClfA, and enterotoxin P). Therefore, the results support the hypothesis that exchange of homologous genes among
S. aureus
genomes can play a role in the evolution of pathogenesis in this species.
Smx Nuclease Is the Major, Low-pH-Inducible Apurinic/Apyrimidinic Endonuclease in
Streptococcus mutans
Authors: Faustoferri Roberta C. , Hahn Kristina , Weiss Kellie , Quivey Robert G. 
Published: April 2005

ABSTRACT

The causative agent of dental caries in humans,
Streptococcus mutans
, outcompetes other bacterial species in the oral cavity and causes disease by surviving acidic conditions in dental plaque. We have previously reported that the low-pH survival strategy of
S. mutans
includes the ability to induce a DNA repair system that appears to involve an enzyme with exonuclease functions (K. Hahn, R. C. Faustoferri, and R. G. Quivey, Jr., Mol. Microbiol 31:1489-1498, 1999). Here, we report overexpression of the
S. mutans
apurinic/apyrimidinic (AP) endonuclease, Smx, in
Escherichia coli
; initial characterization of its enzymatic activity; and analysis of an
smx
mutant strain of
S. mutans
. Insertional inactivation of the
smx
gene eliminates the low-pH-inducible exonuclease activity previously reported. In addition, loss of Smx activity renders the mutant strain sensitive to hydrogen peroxide treatment but relatively unaffected by acid-mediated damage or near-UV irradiation. The
smx
strain of
S. mutans
was highly sensitive to the combination of iron and hydrogen peroxide, indicating the likely production of hydroxyl radical by Fenton chemistry with concomitant formation of AP sites that are normally processed by the wild-type allele. Smx activity was sufficiently expressed in
E. coli
to protect an
xth
mutant strain from the effects of hydrogen peroxide treatment. The data indicate that
S. mutans
expresses an inducible, class II-like AP endonuclease, encoded by the
smx
gene, that exhibits exonucleolytic activity and is regulated as part of the acid-adaptive response of the organism. Smx is likely the primary, if not the sole, AP endonuclease induced during growth at low pH values.
Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity
Authors: Redick Sambra D. , Stricker Jesse , Briscoe Gina , Erickson Harold P. 
Published: April 2005

ABSTRACT

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 μM, in contrast to the high GTPase activity of wild-type FtsZ above 1 μM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 μM

K
d

for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.
cse
, a Chimeric and Variable Gene, Encodes an Extracellular Protein Involved in Cellular Segregation in
Streptococcus thermophilus
Authors: Borges Frédéric , Layec Séverine , Thibessard Annabelle , Fernandez Annabelle , Gintz Brigitte , Hols Pascal , Decaris Bernard , Leblond-Bourget Nathalie 
Published: April 2005

ABSTRACT

The isolation of a
Streptococcus thermophilus
CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the
cse
gene (for cellular segregation). The N terminus of Cse exhibits high similarity to
Streptococcus agalactiae
surface immunogenic protein (SIP), while its C terminus exhibits high similarity to
S. thermophilus
PcsB. In CNRZ368, deletion of the entire
cse
open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Δ
cse
mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of
cse
from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311
cse
was exchanged with that of CNRZ368
cse
. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the
cse
locus suggests that the chimeric organization of
cse
results from insertion of a duplicated sequence deriving from the
pcsB
3′ end into an ancestral
sip
gene. Thus, the
cse
locus illustrates the module-shuffling mechanism of bacterial gene evolution.
Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate
Authors: Mahapatra Sebabrata , Yagi Tetsuya , Belisle John T. , Espinosa Benjamin J. , Hill Preston J. , McNeil Michael R. , Brennan Patrick J. , Crick Dean C. 
Published: April 2005

ABSTRACT

Structural analysis of compounds identified as lipid I and II from
Mycobacterium smegmatis
demonstrated that the lipid moiety is decaprenyl phosphate; thus,
M. smegmatis
is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly
N-
acetylmuramyl-
l
-alanine-
d
-glutamate-
meso
-diaminopimelic acid-
d
-alanyl-
d
-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as
N
-acetylmuramic acid,
N-
glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the
meso
-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal
d
-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of
M. smegmatis
are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.
Participation of 3′-to-5′ Exoribonucleases in the Turnover of
Bacillus subtilis
mRNA
Authors: Oussenko Irina A. , Abe Teppei , Ujiie Hiromi , Muto Akira , Bechhofer David H. 
Published: April 2005

ABSTRACT

Four 3′-to-5′ exoribonucleases have been identified in
Bacillus subtilis
: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5′ ends were at the transcriptional start site and whose 3′ ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of
rpsO
mRNA decay showed that decay intermediates accumulated as the result of a block to 3′-to-5′ processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA
trans
translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.
Catabolite Repression of the Propionate Catabolic Genes inEscherichia coliandSalmonella enterica: Evidence for Involvement of the Cyclic AMP Receptor Protein
Authors: Lee Sung Kuk , Newman Jack D. , Keasling Jay D. 
Published: April 2005

ABSTRACTPrevious studies withSalmonella entericaserovar Typhimurium LT2 demonstrated that transcriptional activation of theprpBCDEoperon requires the function of transcription factor PrpR, sigma-54, and IHF. In this study, we found that transcription from theprpBCDEandprpRpromoters was down-regulated by the addition of glucose or glycerol, indicating that these genes may be regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Targeted mutagenesis of a putative CRP-binding site in the promoter region betweenprpRandprpBCDEsuggested that these genes are under the control of CRP. Furthermore, cells with defects incyaorcrpexhibited reduced transcriptional activation ofprpRandprpBCDEinEscherichia coli. These results demonstrate that propionate metabolism is subject to catabolite repression by the global transcriptional regulator CRP and that this regulation is effected through control of both the regulator geneprpRand theprpBCDEoperon itself. The unique properties of the regulation of these two divergent promoters may have important implications for mechanisms of CRP-dependent catabolite repression acting in conjunction with a member of the sigma-54 family of transcriptional activators.
Antimutator Role of DNA Glycosylase MutY in Pathogenic
Neisseria
Species
Authors: Davidsen T. , Bjørås M. , Seeberg E. C. , Tønjum T. 
Published: April 2005

ABSTRACT

Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of
Neisseria meningitidis
, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in
Escherichia coli
exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2′-deoxyguanosine and A:C mismatches. Expression in
E. coli
fully suppressed the elevated spontaneous mutation rate found in the
E. coli mutY
mutant. An assessment of MutY activity in lysates of neisserial wild-type and
mutY
mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC
mutY
mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in
rpoB
conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC→AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.
Cytochrome Oxidase Deficiency Protects
Escherichia coli
from Cell Death but Not from Filamentation Due to Thymine Deficiency or DNA Polymerase Inactivation
Authors: Strauss Bernard , Kelly Kemba , Ekiert Damian 
Published: April 2005

ABSTRACT

Temperature-sensitive DNA polymerase mutants (
dnaE
) are protected from cell death on incubation at nonpermissive temperature by mutation in the
cydA
gene controlling cytochrome
bd
oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The
cydA
mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both
dnaE
and
Δthy
mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the
dnaE cydA
double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of
cydA
at high temperature is itself suppressed by overexpression of cytochrome
bo3
, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the
cydA
protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome
bd
oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage.
Polarity of Enteropathogenic
Escherichia coli
EspA Filament Assembly and Protein Secretion
Authors: Crepin Valérie F. , Shaw Robert , Abe Cecilia M. , Knutton Stuart , Frankel Gad 
Published: April 2005

ABSTRACT

Type III secretion systems (TTSS) are sophisticated macromolecular structures that play an imperative role in bacterial infections and human disease. The TTSS needle complex is conserved among bacterial pathogens and shows broad similarity to the flagellar basal body. However, the TTSS of enteropathogenic and enterohemorrhagic
Escherichia coli
, two important human enteric pathogens, is unique in that it has a ∼12-nm-diameter filamentous extension to the needle that is composed of the secreted translocator protein EspA. EspA filaments and flagellar structures have very similar helical symmetry parameters. In this study we investigated EspA filament assembly and the delivery of effector proteins across the bacterial cell wall. We show that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament. Moreover, EspA filament length is modulated by the availability of intracellular EspA subunits. Finally, we provide direct evidence that EspA filaments are hollow conduits through which effector proteins are delivered to the extremity of the bacterial cell (and subsequently into the host cell).
Clonal Distribution and Phase-Variable Expression of a Major Histocompatibility Complex Analogue Protein in
Staphylococcus aureus
Authors: Buckling Angus , Neilson James , Lindsay Jodi , ffrench-Constant Richard , Enright Mark , Day Nicholas , Massey Ruth C. 
Published: April 2005

ABSTRACT

The
mapW
gene of
Staphylococcus aureus
strain N315 contains a poly(A) tract which truncates translation of the protein. This study demonstrates that
mapW
is an allelic variant of the
map/eap
genes found in other strains and that the variation in the length of this poly(A) tract suggests that it is a contingency locus.
Positive Selection for Loss-of-Function
tat
Mutations Identifies Critical Residues Required for TatA Activity
Authors: Hicks Matthew G. , Lee Philip A. , Georgiou George , Berks Ben C. , Palmer Tracy 
Published: April 2005

ABSTRACT

The Tat system, found in the cytoplasmic membrane of many bacteria, is a general export pathway for folded proteins. Here we describe the development of a method, based on the transport of chloramphenicol acetyltransferase, that allows positive selection of mutants defective in Tat function. We have demonstrated the utility of this method by selecting novel loss-of-function alleles of
tatA
from a pool of random
tatA
mutations. Most of the mutations that were isolated fall in the amphipathic region of TatA, emphasizing the pivotal role that this part of the protein plays in TatA function.
Assessing automobile head restraint positioning in Portland, Oregon
Authors: Young A L , Ragel B T , Su E , Mann C N , Frank E H 
Published: April 2005

Objective:Automobile head restraints, when used properly, have been shown to decrease the incidence and severity of whiplash injuries to the neck. Before the development of a public campaign on proper head restraint positioning, the authors assessed head restraint positioning and public understanding.Design:Over a one month period, the position of the vehicle head restraint of drivers was observed in moving cars in the city of Portland, Oregon (population 530 000). Optimal position was defined as having the head restraint above the ears with the back of the head touching the head restraint. A questionnaire on head restraint understanding was administered to people during jury service.Results:Of the 4287 drivers observed, 1% (n = 30) had no head restraint on their seat, 4% (n = 158) had a fixed head restraint, and 95% (n = 4099) had an adjustable head restraint. Among the fixed head restraints, 21% (33/158) were positioned optimally with no horizontal gap. Among the adjustable head restraints, only 7% (280/4099) had optimal head restraint positioning. Overall, 93% (3974/4287) of all head restraints observed were suboptimally positioned. Seventy five percent (38/51) of polled Portland residents identified safety as the primary head restraint function.Conclusion:Ninety three percent of all head restraints observed were suboptimally positioned. Fixed head restraints were three times more likely to be in optimal position than adjustable head restraints (21%v7%). Most polled Portland residents understood the proper function and positioning of head restraints. This discrepancy between actual practice and understanding should be addressed with public education and manufacturer design changes.