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The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli. Richard A. Lease, 2004.DsrA RNA is a small [87-nucleotide] regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translationand turnover of specific mRNAs . Two targets of DsrA regulationare RpoS, the stationary-phase and stress response sigma factor[  s],
and H-NS, a histone-like nucleoid protein and global transcription
repressor . Genes regulated globally by RpoS and H-NS includestress
response proteins and virulence factors for pathogenicE . coli .
Here, by using transcription profiling via DNA arrays,we have
identified genes induced by DsrA . Steady-state levelsof mRNAs from
many genes increased with DsrA overproduction,including multiple
acid resistance genes of E . coli . Quantitativeprimer
extension analysis verified the induction of individualacid
resistance genes in the hdeAB, gadAX, and gadBC operons.
E . coli K-12 strains, as well as pathogenic E . coli O157:H7,
exhibited compromised acid resistance in dsrA mutants . Conversely,
overproduction of DsrA from a plasmid rendered the acid-sensitive
dsrA mutant extremely acid resistant . Thus, DsrA RNA plays a
regulatory role in acid resistance . Whether DsrA targets acid
resistance genes directly by base pairing or indirectly via
perturbation of RpoS and/or H-NS is not known, but in eitherevent,
our results suggest that DsrA RNA may enhance the virulenceof
pathogenic E . coli.
Identification of Inducers of the Yersinia enterocolitica Phage Shock Protein System and Comparison to the Regulation of the RpoE and Cpx Extracytoplasmic Stress Responses. Michelle E. Maxson, 2004.Known inducers of the phage shock protein (Psp) system suggest that it is an extracytoplasmic stress response, as are the well-studied RpoE and Cpx systems . However, a random approach to identify conditions and proteins that induce the Psp system has not been attempted . It is also unknown whether the proteins or mutations that induce Psp are specific or if they also activate the RpoE and Cpx systems . This study addressed these issues for the Yersinia enterocolitica Psp system . Random transposon mutagenesis identified null mutations and overexpression mutations that increase  (pspA-lacZ)
operon fusion expression . The results suggest that Psp may respond
exclusively to extracytoplasmic stress . Null mutations affected
glucosamine-6-phosphate synthetase (glmS), which plays a role
in cell envelope biosynthesis, and the F0F1 ATPase (atp
operon) . The screen also revealed that in addition to several
secretins, the overexpression of three novel putative inner membrane
proteins (IMPs) induced the Psp response . We also compared induction
of the Y . enterocolitica Psp, RpoE, and Cpx responses .
Overexpression of secretins or the three IMPs or the presence of an
atpB null mutation only induced the Psp response . Similarly,
known inducers of the RpoE and Cpx responses did not significantly
induce the Psp response . Only the glmS null mutation induced
all three responses . Therefore, Psp is induced distinctly from the
RpoE and Cpx systems . The specific IMP inducers may be valuable tools
to probe specific signal transduction events of the Psp response
in future studies .
Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods. Suleyman Yildirim, 2004.Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis . However, the health hazards posed by L . monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods . In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L . monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI . These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L . monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America . This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis . Toxic Waste Disposal in Escherichia coli. Robert B. Helling, 2002.About 10% of the nalidixic acid-resistant (Nalr) mutants in a transposition-induced library exhibited a growth factor requirement as the result of cysH, icdA, metE, or purB mutation . Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role . Transcription of acrAB was increased in each type of Nalr mutant . In the icdA and purB mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump . The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism . Substrate Specificity of the RND-Type Multidrug Efflux Pumps AcrB and AcrD of Escherichia coli Is Determined Predominately by Two Large Periplasmic Loops. Christopher A. Elkins, 2002.AcrAB-TolC is a constitutively expressed, tripartite efflux transporter complex that functions as the primary resistance mechanism to lipophilic drugs, dyes, detergents, and bile acids in Escherichia coli . TolC is an outer membrane channel, and AcrA is an elongated lipoprotein that is hypothesized to span the periplasm and coordinate efflux of such substrates by AcrB and TolC . AcrD is an efflux transporter of E . coli that provides resistance to aminoglycosides as well as to a limited range of amphiphilic agents, such as bile acids, novobiocin, and fusidic acid . AcrB and AcrD belong to the resistance nodulation division superfamily and share a similar topology, which includes a pair of large periplasmic loops containing more than 300 amino acid residues each . We used this knowledge to test several plasmid-encoded chimeric constructs of acrD and acrB for substrate specificity in a marR1  acrB
acrD host . AcrD chimeras were constructed in which the large, periplasmic loops between transmembrane domains 1 and 2 and 7 and 8 were replaced with the corresponding loops of AcrB . Such constructs provided resistance to AcrB substrates at levels similar to native AcrB . Conversely, AcrB chimeras containing both loops of AcrD conferred resistance only to the typical substrates of AcrD . These results cannot be explained by simply assuming that AcrD, not hitherto known to interact with AcrA, acquired this ability by the introduction of the loop regions of AcrB, because (i) both AcrD and AcrA were found, in this study, to be required for the efflux of amphiphilic substrates, and (ii) chemical cross-linking in intact cells efficiently produced complexes between AcrD and AcrA . Since AcrD can already interact with AcrA, the alterations in substrate range accompanying the exchange of loop regions can only mean that substrate recognition (and presumably binding) is determined largely by the two periplasmic loops .
Products Transcribed from Rearranged rrn Genes of Escherichia coli Can Assemble To Form Functional Ribosomes. Dmitry Zaporojets, 2003.To examine the flexibility of rRNA operons with respect to fundamental organization, transcription, processing, and assembly of ribosomes, operon variations were introduced by a plasmid into an Escherichia coli strain that has deletions of all chromosomal copies of rRNA genes . In the reconstructed operons, a Salmonella intervening sequence (IVS) from 23S helix 45 was introduced into the E . coli 23S gene at the same position . Three different constructs of the E . coli 16S gene were then placed wholly within the IVS sequence, and the 16S gene was deleted from its normal position . The resulting plasmids thus had the normal operon promoters and the leader region followed by the 5' one-third of the 23S gene, the entire 16S gene within the IVS, the last two-thirds of the 23S gene, and the normal end of the operon . The three constructs differed in the amount of 16S leader and spacer regions they contained . Only two of the three constructs, those with redundant leader and spacer antiterminator signals, resulted in viable cultures of the rrn deletion strain . Electron micrographs of the variant operon suggest that the 23S rRNA is made in two separate parts which then must form subassemblies before assembling into a functional 50S subunit . Cells containing only the reshuffled genes were debilitated in their growth properties and ribosome contents . The fact that such out of the ordinary manipulation of rRNA sequences in E . coli is possible paves the way for detailed analysis of ribosome assembly and evolution . A Potential Food-Grade Cloning Vector for Streptococcus thermophilus That Uses Cadmium Resistance as the Selectable Marker. Wing Yee Wong, 2003.A potential food-grade cloning vector, pND919, was constructed and transformed into S . thermophilus ST3-1, a plasmid-free strain . The vector contains DNAs from two different food-approved organisms, Streptococcus thermophilus and Lactococcus lactis . The 5.0-kb pND919 is a derivative of the cloning vector pND918 (9.3 kb) and was constructed by deletion of the 4.3-kb region of pND918 which contained DNA from non-food-approved organisms . pND919 carries a heterologous native cadmium resistance selectable marker from L . lactis M71 and expresses the Cdr phenotype in S . thermophilus transformants . With the S . thermophilus replicon derived from the shuttle vector pND913, pND919 is able to replicate in the two S . thermophilus industrial strains tested, ST3-1 and ST4-1 . Its relatively high retention rate in S . thermophilus further indicates its usefulness as a potential food-grade cloning vector . To our knowledge, this is the first report of a replicative potential food-grade vector for the industrially important organism S . thermophilus .
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