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Archives of Microbiology (v.170, #3)

Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli by B. M. Prüß (pp. 141-146).

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Keywords: Key words FlhD; Acetyl phosphate; OmpR; Flagella; expression; Cell division

Biochemical properties of the products of cytochrome P450 genes (PDA) encoding pisatin demethylase activity in Nectria haematococca by H. L. George; K. D. Hirschi; H. D. VanEtten (pp. 147-154).

Pea plants produce the antibiotic (+)pisatin in response to infection by the fungus Nectria haematococca, which can detoxify pisatin utilizing a cytochrome P450 monooxygenase called pisatin demethylase. Genes (PDA) have been identified that encode different whole-cell Pda phenotypes that can be distinguished by the length of the lag period and the resulting amount of enzyme activity produced: Pda^SH = short lag, high activity; Pda^SM = short lag, moderate activity; and Pda^LL = long lag, low activity. Only the Pda^SH and Pda^SM phenotypes have been correlated with pathogenicity on pea. In this study, we utilize heterologous expression of the PDA ^LL gene PDA6-1 in Aspergillus nidulans to compare the biochemical properties of the product of this gene with the products of the PDA ^SH gene PDA1 expressed in N. haematococca. Preliminary measurements were also done on the PDA ^SM gene PDA5 expressed in N. haematococca. The PDA gene products differed somewhat in their substrate specificity and in their sensitivity to a few cytochrome P450 inhibitors. However, the enzymes produced by PDA6-1 and PDA1 both had low apparent K _m values toward (+)pisatin (< 0.25 μM) and a common high degree of insensitivity to most P450 inhibitors, suggesting similar shared biochemical traits as would be expected of products of a highly homologous gene family. Our results indicate that the different whole-cell phenotypes of N. haematococca are not due to significant differences in the biochemical properties of the gene products and are consistent with recent results that indicate that the phenotypic differences are due to different degrees of expression of the genes.

Keywords: Key words Phytoalexin; Isoflavonoid; Detoxification; Monooxygenase; Fusarium solani; Nectria; haematococca; Pisatin demethylase; Cytochrome P450

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus by T. Itoh; Ken-ichiro Suzuki; Takashi Nakase (pp. 155-161).

Multiple introns were detected in the 16S rRNA gene of newly isolated Thermoproteus species strains IC-033 and IC-061 and Thermoproteus neutrophilus JCM 9278. In the 16S rRNA gene of strain IC-033, five introns of 627, 762, 636, 33, and 682 bp existed after positions 548, 781, 1092, 1205, and 1213 (according to the Escherichia coli numbering system), respectively. Likewise, strain IC-061 possessed 764-, 32-, and 688-bp introns after positions 781, 1205, and 1213, respectively; and T. neutrophilus JCM 9278 had 34- and 663-bp introns after positions 1205 and 1213, respectively. All the introns carried the putative intron core structures consisting of a bulge-helix-bulge motif and a long stable stem. The large introns carried open reading frames containing the LAGLI-DADG-like motifs in their terminal inserts; however, three out of four large introns of strain IC-033 seemed to incur frameshift mutations. Occurrence of introns at the same insertion sites in the three strains would allow tracing of the evolutionary movements of these introns.

Keywords: Key words Archaea; Hyperthermophile; Thermoproteus; Intron; 16S rRNA gene; LAGLI-DADG-like motif

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria by Silke Hein; H. Tran; A. Steinbüchel (pp. 162-170).

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction.

Keywords: Key wordsSynechocystis sp. PCC6803; Biodegradable polyester; Polyhydroxyalkanoic acid; Poly(3-hydroxybutyric acid); PHA-synthase; PhaE; PhaC

Teichoic acid content in different lineages of Staphylococcus aureus NCTC8325 by René Jenni; B. Berger-Bächi (pp. 171-178).

A series of mec transformants of Staphylococcus aureus strain NCTC8325 were analysed for alterations in wall teichoic acid and lipoteichoic acid. Although the methicillin resistance determinant alters the autolytic behaviour of S. aureus, it had no effects on the cellular content, chain length, and alanine substitution of the lipoteichoic acid, or on the wall teichoic acid content and composition. However, independently of the presence or absence of the methicillin resistance determinant, level of methicillin resistance, or autolytic behaviour, a correlation was found between a 25% reduced cell wall phosphate content and either loss of prophages φ11 and 13 or a 30-kb deletion in the chomosmal SmaI-F fragment adjacent to the prophage φ11 attachment site.

Keywords: Key wordsStaphylococcus aureus; Wall teichoic acid; Lipoteichoic acid; Lysogeny; Methicillin resistance

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells by Y. Kimura; Toshiyuki Kojyo; I. Kimura; Masayuki Sato (pp. 179-184).

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development.

Keywords: Key wordsMyxococcus xanthus; Propionyl-CoA; carboxylase; Acetyl-CoA carboxylase; Kinetic constant

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5 by Caterina Signoretto; Marzia Boaretti; P. Canepari (pp. 185-190).

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Keywords: Key words Peptidoglycan; β-Lactam antibiotics; Penicillin resistance; Penicillin binding proteins; Enterococcus faecalis

Heat shock effects on second messenger systems of Neurospora crassa by Andreas Kallies; Gerd Gebauer; L. Rensing (pp. 191-200).

Exposure of growing hyphae of Neurospora crassa to heat shock (44 °C) or ethanol (2.6 M) for 1 h induced a significant increase in the cAMP level, which reached a maximum approximately 2 min after the beginning of treatment and then decreased to control values despite continued heat or ethanol exposure. A 10-s heat shock or a 5-s ethanol shock also resulted in a transient cAMP increase 2 min after the pulse. Heat shock or ethanol treatment led to an increase in the amount of catalytic subunits of the cAMP-dependent protein kinase A in the nucleus almost synchronously with the increase of cAMP in the cytoplasm. The concentration of cGMP decreased a few seconds after the beginning of heat shock (44 °C) or ethanol treatment (2.6 M) to approximately 50% of the control level. Exposure to heat shock (44 °C, 1 h) led to an increase in the amount of inositol phosphates 0.5–2 min after the onset of heat shock. Thereafter, inositol phosphate levels dropped to control values despite continued heat exposure. Incubation of growing hyphae with cAMP or 8-Br-cAMP led to a two- to threefold increase of inositol phosphates 10–300 s after the beginning of incubation. Heat treatment furthermore caused a rapid release of calcium from vacuoles as determined by Fura-2 measurement of the calcium content released from isolated vacuoles. These heat-shock-dependent second messenger changes may play a role in the heat-shock-induced phase shifts of the circadian clock and heat-shock-induced conidiation.

Keywords: Key words Heat shock; Ethanol; cAMP; Protein; kinase A; cGMP; Inositol phosphates; Calcium

Nucleic acid content of Synechococcus spp. during growth in continuous light and light/dark cycles by P. W. Lepp; T. M. Schmidt (pp. 201-207).

Light-limited batch cultures of Synechococcus spp. strains PCC 6301 and WH 8103 exhibited a positive correlation between the specific growth rate and the cellular content of both RNA and DNA. The ratio of RNA to DNA increased with the growth rate in Synechococcus sp. strain WH 8103, as it does in heterotrophic bacteria, but remained constant in Synechococcus sp. strain PCC 6301. The cellular content of nucleic acids decreased during light periods and increased during dark periods in Synechococcus sp. strain PCC 6301 entrained by 12-h light/ 12-h dark (diurnal) cycles. This result was unexpected in light of experiments demonstrating a circadian increase in transcriptional activity during the subjective light periods and a decrease in transcriptional activity during subjective dark periods. The cyclical variation in cellular nucleic acid content during diurnal cycles appears to be in part a function of the timing of cell division and will influence estimates of in situ growth rates or relative abundances of cyanobacteria using oligonucleotide probes complementary to 16S rRNA.

Keywords: Key words Cyanobacteria; Nucleic acid content; Circadian rhythms; Cell division; Ribosomal RNA

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Archives of Microbiology (v.170, #3)