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Transposon-Mediated Linker Insertion Scanning Mutagenesis of the Escherichia coli McrA Endonuclease. Brian P. Anton, 2004.McrA is one of three functions that restrict modified foreign DNA in Escherichia coli K-12, affecting both methylated and hydroxymethylated substrates . We present here the first systematic analysis of the functional organization of McrA by using the GPS-LS insertion scanning system . We collected in-frame insertions of five amino acids at 46 independent locations and C-terminal truncations at 20 independent locations in the McrA protein . Each mutant was assayed for in vivo restriction of both methylated and hydroxymethylated bacteriophage (M.HpaII-modified 
and T4gt, respectively) and for induction of the E . coli
SOS response in the presence of M.HpaII methylation, indicative of
DNA damage . Our findings suggest the presence of an N-terminal
DNA-binding domain and a C-terminal catalytic nuclease domain
connected by a linker region largely tolerant of amino acid
insertions . DNA damage inflicted by a functional C-terminal domain is
required for restriction of phage T4gt . Disruption of the
N-terminal domain abolishes restriction of both substrates .
Surprisingly, truncation mutations that spare the N-terminal domain
do not mediate DNA damage, as measured by SOS induction, but
nevertheless partially restrict M.HpaII-modified
in vivo . We suggest a common explanation for this "restriction
without damage" and a similar observation seen in vivo with McrB, a
component of another of the modified-DNA restriction functions .
Briefly, we propose that unproductive site-specific binding of the
protein to a vulnerable position in the
genome disrupts the phage development program at an early stage . We
also identified a single mutant, carrying an insertion in the
N-terminal domain, which could fully restrict
but did not restrict T4gt at all . This mutant may have a
selective impairment in substrate recognition, distinguishing
methylated from hydroxymethylated substrates . The study shows that
the technically easy insertion scanning method can provide a rich
source of functional information when coupled with effective
phenotype tests .
Quinolone-Resistant Haemophilus influenzae: Determination of Mutant Selection Window for Ciprofloxacin, Garenoxacin, Levofloxacin, and Moxifloxacin. Xinying Li, 2004.Stepwise selection of ciprofloxacin-resistant Haemophilus influenzae mutants produced first-, second-, third-, and fourth-step substitutions in GyrA (S84Y), ParC (S84R), GyrA (D88N), and ParC (E88K), respectively . Successive mutations raised the mutant selection window . The wild-type selection window for garenoxacin, levofloxacin, and moxifloxacin was also measured . New Method for Estimating Bacterial Cell Abundances in Natural Samples by Use of Sublimation. Daniel P. Glavin, 2004.We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples . To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500°C for several seconds under reduced pressure . The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection . Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from  105 to 109 E . coli cell equivalents per gram . For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining .
MxiE Regulates Intracellular Expression of Factors Secreted by the Shigella flexneri 2a Type III Secretion System. Colleen D. Kane, 2002.The mxi-spa locus on the virulence plasmid of Shigella flexneri encodes components of the type III secretion system . mxiE, a gene within this locus, encodes a protein that is homologous to the AraC/XylS family of transcriptional regulators, but currently its role in pathogenesis remains undefined . We characterized the virulence phenotype of a nonpolar mxiE mutant and found that this mutant retained the ability to invade mammalian cells in tissue culture and secrete Ipas (type III effectors required for host cell invasion), although it was less efficient than wild-type Shigella at cell-to-cell spread . Despite its invasive properties in culture, the mxiE mutant was completely avirulent in an animal model . Potential targets for MxiE activation were identified by using promoter-green fluorescent protein fusions, and gene expression was examined under various growth conditions . Six MxiE-regulated genes were discovered: ospB, ospC1, ospE2, ospF, virA, and ipaH9.8 . Notably, activation of these genes only occurred within the intracellular environment of the host and not during growth at 37°C in liquid culture . Interestingly, all of the MxiE-regulated proteins previously have been shown to be secreted through the type III secretion system and are putative virulence factors . Our findings suggest that some of these Osp proteins may be involved in postinvasion events related to virulence . Since bacterial pathogens adapt to multiple environments during the course of infecting a host, we propose that Shigella evolved a mechanism to take advantage of a unique intracellular cue, which is mediated through MxiE, to express proteins when the organism reaches the eukaryotic cytosol . Fingerprinting Microbial Assemblages from the Oxic/Anoxic Chemocline of the Black Sea. Costantino Vetriani, 2003.Biomass samples from the Black Sea collected in 1988 were analyzed for SSU genes from Bacteria and Archaea after 10 years of storage at -80°C . Both clonal libraries and direct fingerprinting by terminal restriction fragment length polymorphism (T-RFLP) analyses were used to assess the microbial community . Uniform and discrete depth distributions of different SSU phylotypes were observed . However, most recombinant clones were not restricted to a specific depth in the water column, and many of the major T-RFLP peaks remain uncharacterized . Of the clones obtained, an  -Proteobacteria and a Pseudoalteromonas-like clone accounted for major peaks in the fingerprint, while deeply branching lineages of
 - and
 -Proteobacteria were associated with smaller peaks . Additionally, members were found among both the
 -Proteobacteria related to sulfate reducers and the Archaea related to phylotypes from the ANME groups that anaerobically oxidize methane .
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