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Archives of Microbiology (v.178, #3)
No Title by Brigitte Berger-Bächi; Susanne Rohrer (pp. 165-171).
Methicillin resistance in staphylococci is due to an acquired penicillin-binding protein, PBP2′ (PBP2a). This additional PBP, encoded by mecA, confers an intrinsic resistance to all β-lactams and their derivatives. Resistance levels in methicillin-resistant Staphylococcus aureus (MRSA) depend on efficient PBP2′ production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired mecA, resistance levels range from phenotypically susceptible to highly resistant. Characteristic for most MRSA is the heterogeneous expression of resistance, which is due to the segregation of a more highly resistant subpopulation upon challenge with methicillin. Maximal expression of resistance by PBP2′ requires the efficient and correct synthesis of the peptidoglycan precursor. Genes involved in cell-wall precursor formation and turnover, regulation, transport, and signal transduction may determine the level of resistance that is expressed. At this stage, however, there is no information available on the functionality or efficacy of such factors in clinical isolates in relation to methicillin resistance levels.
Keywords: aux fem Staphylococcus aureus mecA mecR1 mecI Methicillin resistance Penicillinase Regulation SCCmec
No Title by Kyoko Yasumura; Kazuei Igarashi; Yoshimi Kakinuma (pp. 172-179).
The Enterococcus hirae ntp operon encodes all subunits of the vacuolar-type ATPase (V-ATPase), which transports Na+ or Li+. This operon is expressed preferentially in response to Na+, but not to Li+. Deletion analysis of the ntp promoter region in plasmids indicated that the AT-tract between –198 and –132 is required for Na+-specific transcriptional regulation. In addition, lithium-tolerant (LTR) mutants were isolated in which functional V-ATPase levels were high even in Na+-depleted medium. Western blot and Northern blot experiments revealed an increase in basal Na+-independent transcription in one of the mutants (LTR1). The nucleotide sequences of the ntp promoter region of the LTR mutants showed mutational conversion of single base-pairs between positions –23 and +1. Na+-independent expression of a reporter gene linked to the ntp promoter in plasmids was elevated by base substitutions at –23 to +1, and promoter activity induced by these base substitutions was lost by deletion of the region between –198 and –132. These results suggest that the AT-tract between –198 and –132 is indispensable for transcription of the ntp operon.
Keywords: V-ATPase Operon Expression Promoter Enterococcus hirae
No Title by Magdy Mohamed; Wael Ismail; Johann Heider; Georg Fuchs (pp. 180-192).
The aerobic metabolism of phenylacetic acid (PA) and 4-hydroxyphenylacetic acid (4-OHPA) was investigated in the β-proteobacterium Azoarcus evansii. Evidence for the existence of two independent catabolic pathways for PA and 4-OHPA is presented. 4-OHPA metabolism involves the formation of 2,5-dihydroxyphenylacetate (homogentisate) and maleylacetoacetate catalyzed by specifically induced 4-OHPA 1-monooxygenase and homogentisate 1,2-dioxygenase. The metabolism of PA starts by its activation to phenylacetyl-CoA (PA-CoA) via an aerobically induced phenylacetate-coenzyme A ligase. Phenylalanine (Phe) aerobic metabolism in this bacterium proceeds also via PA and PA-CoA. Whole cells of A. evansii transformed [1-14C]PA to 14C-phenylacetyl-CoA and subsequently to a number of unknown labeled products, which were also observed in PA-degrading bacteria from different phylogenetic groups, i.e. Escherichia coli, Rhodopseudomonas palustris and Bacillus stearothermophilus. A chromosomal region from A. evansii of 11.5 kb containing a cluster of 11 phenylacetic acid catabolic (paa) genes (paaYZGHIKABCDE) was sequenced and characterized. The derived gene products were similar to the characterized putative gene products involved in PA catabolism in E. coli and Pseudomonas putida and to other putative PA catabolic gene products of diverse bacteria. RT-PCR analysis of the paa genes of A. evansii growing aerobically with PA showed a probable organization of the paa genes in three operons. The similarity of the PA metabolic products pattern and of gene sequences suggests a common aerobic bacterial PA pathway.
Keywords: Phenylacetic acids Aerobic metabolism Azoarcus evansii Monooxygenase Dioxygenase paa gene cluster
No Title by Rachel A. Larsen; Marlena M. Wilson; Adam M. Guss; William W. Metcalf (pp. 193-201).
A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1×10–3 per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10–3. Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.
Keywords: Transposon Tn5 Mutagenesis Carotenoid Xanthobacter
No Title by Stephan Braatsch; Torsten Krafft; Jörg Simon; Roland Gross; Oliver Klimmek; Achim Kröger (pp. 202-207).
Wolinella succinogenes grows by polysulfide respiration with formate or hydrogen as electron donor. Polysulfide reduction is catalyzed by the membrane-bound polysulfide reductase complex encoded by the psrABC operon. An open reading frame, designated psrR, was found in close proximity upstream of the psr operon, but oriented in the opposite direction. The deduced amino acid sequence of PsrR is similar to those of transcriptional regulators of the AraC family and includes all typical features. Polysulfide reductase is not detectable in a ΔpsrR deletion mutant of W. succinogenes. Mutant cells grown with fumarate as terminal electron acceptor did not catalyze polysulfide reduction with formate or hydrogen, in contrast to the wild-type strain. The phenotype of W. succinogenes wild-type cells was restored by genomic complementation of W. succinogenes ΔpsrR. The results suggest that the gene product of psrR is involved in the regulation of polysulfide reductase synthesis.
Keywords: Transcriptional regulator AraC family Polysulfide reduction Wolinella succinogenes Anaerobic respiration
No Title by Diana Di Gioia; Claudia Barberio; Sonia Spagnesi; Leonardo Marchetti; Fabio Fava (pp. 208-217).
Seven aerobic bacterial strains capable of degrading several of the monocyclic aromatic compounds occurring in the phenolic fraction of olive-mill wastewaters (OMWs) were isolated from an Italian OMW. The results of the 16S rDNA restriction analysis evidenced that these strains are distributed among four different groups. One strain of each group was taxonomically characterized by sequencing the amplified 16S rDNA, and the four strains were assigned to the genera Comamonas (strain AV1A), Ralstonia (strain AV5BG), Pseudomonas (strain AV2A) and Sphingomonas (strain AV6C). The four strains, when checked for the ability to degrade nine monocyclic aromatic compounds abundant in OMWs, were found to significantly metabolize five to eight of them, both as resting cells and growing cells. Specific enzyme analyses of the same selected strains showed: (1) the occurrence of O-demethylating activities towards four methoxylated mono-aromatic acids in three of the four studied strains (strains AV1A, AV5BG and AV6C), (2) ring-cleavage activity towards protocatechuic acid in all of the strains, and (3) a ring-cleavage activity towards catechol in strain AV6C. The isolates described here exhibit a biodegradation potential towards monocyclic aromatic compounds of OMWs that is markedly broader and higher than that displayed by other aerobic bacteria described previously. These features make them excellent candidates for removing the low-molecular-weight phenolic compounds persisting in the effluent following anaerobic digestion of OMWs.
Keywords: Olive-mill wastewater Biodegradation Comamonas Ralstonia Pseudomonas Sphingomonas Hydroxylated and methoxylated benzoic acids Hydroxylated phenyl acetic acids Hydroxylated and methoxylated phenyl propenoic acids
No Title by Gilbert Lamothe; Laure Jolly; Beat Mollet; Francesca Stingele (pp. 218-228).
Lactobacillus delbrueckii subsp. bulgaricus produces exopolysaccharides (EPSs), which play a role in the rheological properties of fermented food products. Lb. bulgaricus Lfi5 produces a high-molecular-weight EPS composed of galactose, glucose, and rhamnose in the molar ratio 5:1:1. An 18-kb DNA region containing 14 genes, designated epsA to epsN, was isolated by genomic DNA library screening and inverted PCR. The predicted gene products are homologous to proteins involved in the biosynthesis of other bacterial polysaccharides and the genetic organization was found to be similar to that of other eps clusters from lactic acid bacteria. Transcriptional analysis revealed that the 14 eps genes are co-ordinately expressed and transcribed as a single mRNA of 15–16 kb. The transcription start site of the promoter was mapped upstream of the first gene, epsA. Genes encoding glycosyltranferases were further studied by heterologous expression and functional assays. We showed that the epsE gene product is a phospho-glucosyltransferase initiating the biosynthesis of EPS. Heterologous expression of epsE in a Lactococcus lactis epsD mutant restored EPS production, demonstrating its role and importance in EPS biosynthesis. Functional assays of other glycosyltransferases allowed their sugar specificity to be elucidated and an overall biosynthetic pathway for EPS synthesis by Lb. bulgaricus to be proposed.
Keywords: Lactic acid bacteria Lactobacillus delbrueckii bulgaricus eps gene cluster Exopolysaccharide Glycosyltransferase
No Title by Marcel T. van der Meer; Stefan Schouten; Satoshi Hanada; Ellen C. Hopmans; Jaap S. Damsté; David M. Ward (pp. 229-237).
The lipid composition of Roseiflexus castenholzii, a thermophilic filamentous phototrophic bacterium related to uncultivated filamentous phototrophic bacteria that predominate in hot spring microbial mats, is reported. R. castenholzii lipid extracts were dominated by components characterized by alkane-1-ol-2-alkanoate moieties glycosidically bonded to a C6 sugar. Similar fatty glycosides, with an additional fatty acid esterified, were detected by HPLC-MS. R. castenholzii also produces a suite of wax esters ranging from 37 to 40 carbon atoms in length. In lipid extracts from two nonsulfidic hot spring microbial mats, similar alkane-1,2-diol-based lipids were detected in minor amounts. R. castenholzii lipids are compared to lipids of mats and other thermophilic mat isolates.
Keywords: Roseiflexus castenholzii Chloroflexus Hot spring microbial mat Alkane-1,2-diol Lipid