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AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus. Ruifeng Yang, 2004.The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait . MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation . Genetic studies showed thataglZ is part of the A motility system, because disruption ordeletion of aglZ abolished movement of isolated cells and aglZsglK double mutants were nonmotile . The aglZ gene encodes a153-kDa protein that interacts with purified MglA in vitro.The N terminus of AglZ shows similarity to the receiver domainof two-component response regulator proteins, while the C terminuscontains heptad repeats characteristic of coiled-coil proteins,such as myosin . Consistent with this motif, expression of AglZin Escherichia coli resulted in production of striated latticestructures . Similar to the myosin heavy chain, the purifiedC-terminal coiled-coil domain of AglZ forms filament structuresin vitro. The YopD Translocator of Yersinia pseudotuberculosis Is a Multifunctional Protein Comprised of Discrete Domains. Jan Olsson, 2004.To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells . YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse . These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH . To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains . We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control . In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified . Significantly, other domains were found to be important for effector microinjection but not for pore formation . Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation . Recognition of this modular YopD domain structure provides important insights into the function of YopD . Northern, Morphological, and Fermentation Analysis of spo0A Inactivation and Overexpression in Clostridium acetobutylicum ATCC 824. Latonia M. Harris, 2002.The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain [824(pMPSOA)] . SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration—a technique never used before with solventogenic clostridia . SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain . After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate . SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores . No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited . Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B . subtilis. Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains . Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control . These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production . Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production . Bex, the Bacillus subtilis Homolog of the Essential Escherichia coli GTPase Era, Is Required for Normal Cell Division and Spore Formation. Natalie Minkovsky, 2002.The Bacillus subtilis bex gene complemented the defect in an Escherichia coli era mutant . The Bex protein showed 39% identity and 67% similarity to the E . coli Era GTPase . In contrast to era, bex was not essential in all strains . bex mutant cells were elongated and filled with diffuse nucleoid material . They grew slowly and exhibited severely impaired spore formation . Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation. Shannon A. Carroll, 2003.A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here . Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L . monocytogenes-specific monoclonal antibody EM-7G1 . MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain . Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus . An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth . Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain . Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L . monocytogenes . Bacillus subtilis ResD Induces Expression of the Potential Regulatory Genes yclJK upon Oxygen Limitation. Elisabeth Härtig, 2004.Transcription of the yclJK operon, which encodes a potential two-component regulatory system, is activated in response to oxygen limitation in Bacillus subtilis . Northern blot analysis and assays of yclJ-lacZ reporter gene fusion activity revealed that the anaerobic induction is dependent on another two-component signal transduction system encoded by resDE . ResDE was previously shown to be required for the induction of anaerobic energy metabolism . Electrophoretic mobility shift assays and DNase I footprinting experiments showed that the response regulator ResD binds specifically to the yclJK regulatory region upstream of the transcriptional start site . In vitro transcription experiments demonstrated that ResD is sufficient to activate yclJ transcription . The phosphorylation of ResD by its sensor kinase, ResE, highly stimulates its activity as a transcriptional activator . Multiple nucleotide substitutions in the ResD binding regions of the yclJ promoter abolished ResD binding in vitro and prevented the anaerobic induction of yclJK in vivo . A weight matrix for the ResD binding site was defined by a bioinformatic approach . The results obtained suggest the existence of a new branch of the complex regulatory system employed for the adaptation of B . subtilis to anaerobic growth conditions . Association of Borrelia garinii and B . valaisiana with Songbirds in Slovakia. Klára Hanincová, 2003.In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks . In most parts of Central Europe, B . afzelii, B . garinii, and B . valaisiana are the most frequent species, whereas B . burgdorferi sensu stricto, B . bissettii, and B . lusitaniae are rare . Previously, it has been shown that B . afzelii is associated with European rodents . Therefore, the aim of this study was to identify reservoir hosts of B . garinii and B . valaisiana in Slovakia . Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks . Questing I . ricinus ticks were collected in the same region . Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing . Three of the 17 captured songbird species were infested with spirochete-infected ticks . Spirochetes in ticks that had fed on birds were genotyped as B . garinii and B . valaisiana, whereas questing ticks were infected with B . afzelii, B . garinii, and B . valaisiana . Furthermore, identical ospA alleles of B . garinii were found in ticks that had fed on the birds and in questing ticks . The data show that songbirds are reservoir hosts of B . garinii and B . valaisiana but not of B . afzelii . This and previous studies confirm that B . burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes .
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