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Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264 . Mauricio G. Martín, 2004.Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex . In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region . Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization . The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb . This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG) . We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs . Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI . This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase . It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions . Functional Analysis of Fructosyl-Amino Acid Oxidases of Aspergillus oryzae . Shin-ichi Akazawa, 2004.Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40 . N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein . FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward N  -fructosyl N -Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val . The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set . The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes . The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active . FAOAo2 disruptant was obtained by using ptrA as a selective marker . Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain
 faoAo2 did not grow . Addition of glucose or (NH4)2SO4 to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene . Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO2 as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase . These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A . oryzae .
Biogeography, Evolution, and Diversity of Epibionts in Phototrophic Consortia . Jens Glaeser, 2004.Motile phototrophic consortia are highly regular associations in which numerous cells of green sulfur bacteria surround a flagellated colorless ß-proteobacterium in the center . To date, seven different morphological types of such consortia have been described . In addition, two immotile associations involving green sulfur bacteria are known . By employing a culture-independent approach, different types of phototrophic consortia were mechanically isolated by micromanipulation from 14 freshwater environments, and partial 16S rRNA gene sequences of the green sulfur bacterial epibionts were determined . In the majority of the lakes investigated, different types of phototrophic consortia were found to co-occur . In all cases, phototrophic consortia with the same morphology from the same habitat contained only a single epibiont phylotype . However, morphologically indistinguishable phototrophic consortia collected from different lakes contained different epibionts . Overall, 19 different types of epibionts were detected in the present study . Whereas the epibionts within one geographic region were very similar (Dice coefficient, 0.582), only two types of epibionts were found to occur on both the European and North American continents (Dice coefficient, 0.190) . None of the epibiont 16S rRNA gene sequences have been detected so far in free-living green sulfur bacteria, suggesting that the interaction between epibionts and chemotrophic bacteria in the phototrophic consortia is an obligate interaction . Based on our phylogenetic analysis, the epibiont sequences are not monophyletic . Thus, the ability to form symbiotic associations either arose independently from different ancestors or was present in a common ancestor prior to the radiation of green sulfur bacteria and the transition to the free-living state in independent lineages . The present study thus demonstrates that there is great diversity and nonrandom geographical distribution of phototrophic consortia in the natural environment . Regulation of the Streptomyces coelicolor Calcium-Dependent Antibiotic by absA, Encoding a Cluster-Linked Two-Component System . N. Jamie Ryding, 2002.The Streptomyces coelicolor absA two-component system was initially identified through analysis of mutations in the sensor kinase absA1 that caused inhibition of all four antibiotics synthesized by this strain . Previous genetic analysis had suggested that the phosphorylated form of AbsA2 acted as a negative regulator of antibiotic biosynthesis in S . coelicolor (T . B . Anderson, P . Brian, and W . C . Champness, Mol . Microbiol . 39:553–566, 2001) . Genomic sequence data subsequently provided by the Sanger Centre (Cambridge, United Kingdom) revealed that absA was located within the gene cluster for production of one of the four antibiotics, calcium-dependent antibiotic (CDA) . In this paper we have identified numerous transcriptional start sites within the CDA cluster and have shown that the original antibiotic-negative mutants used to identify absA exhibit a stronger negative regulation of promoters upstream of the proposed CDA biosynthetic genes than of promoters in the clusters responsible for production of actinorhodin and undecylprodigiosin . The same antibiotic-negative mutants also showed an increase in transcription from a promoter divergent to that of absA, upstream of a putative ABC transporter, in addition to an increase in transcription of absA itself . Interestingly, the negative regulation of the biosynthetic transcripts did not appear to be mediated by transcriptional regulation of cdaR (a gene encoding a homolog of the pathway-specific regulators of the act and red clusters) or by any other recognizable transcriptional regulator associated with the cluster . The role of absA in regulating the expression of the diverse antibiotic biosynthesis clusters in the genome is discussed in light of its location in the cda cluster . Polar Localization of CheA2 in Rhodobacter sphaeroides Requires Specific Che Homologs . Angela C. Martin, 2003.Rhodobacter sphaeroides is a motile bacterium that has multiple chemotaxis genes organized predominantly in three major operons (cheOp1, cheOp2, and cheOp3) . The chemoreceptor proteins are clustered at two distinct locations, the cell poles and in one or more cytoplasmic clusters . One intriguing possibility is that the physically distinct chemoreceptor clusters are each composed of a defined subset of specific chemotaxis proteins, including the chemoreceptors themselves plus specific CheW and CheA proteins . Here we report the subcellular localization of one such protein, CheA2, under aerobic and photoheterotrophic growth conditions . CheA2 is predominantly clustered and localized at the cell poles under both growth conditions . Furthermore, its localization is dependent upon one or more genes in cheOp2 but not those of cheOp1 or cheOp3 . In E . coli, the polar localization of CheA depends upon CheW . The R . sphaeroides cheOp2 contains two cheW genes . Interestingly, CheW2 is required under both aerobic and photoheterotrophic conditions, whereas CheW3 is not required under aerobic conditions but appears to play a modest role under photoheterotrophic conditions . This suggests that R . sphaeroides contains at least two distinct chemotaxis complexes, possibly composed of proteins dedicated for each subcellular location . Furthermore, the composition of these spatially distinct complexes may change under different growth conditions . In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor . Harald Nothaft, 2003.HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria . To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted . The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected . Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 µM . Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity . Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene . Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources . Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated . In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation . However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal . This study provides the first molecular genetic evidence of a physiological role of the PTS in S . coelicolor .
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