- Volume 142, Issue 5, 1996

Actin has been described in all eukaryotic cells as the major microfilament cytoskeletal protein. Although prokaryotic cells do not have a cytoskeleton, proteins related to the latter have been found in different prokaryotic species. We have found prokaryotic actin-related proteins in the enterobacterium Escherichia coli and in the cyanobacteria Anabaena cylindrica and Anabaena variabilis. They were identified by the following criteria: (1) by cross-reaction with a fluorescent conjugated anti-actin (rat-brain) mAb by Western blot analysis (in total cellular extracts); (2) specific binding of acetone powder and soluble cellular extracts to DNase 1; and (3) specific binding of cells and total cellular extracts to phalloidin. In E. coli, specific binding of phalloidin labelled with rhodamine to cells was detected by spectrofluorometry. In total cellular extracts, three bands of 60, 43 and 35 kDa were weakly recognized by the mAb by Western blot analysis; this recognition increased when phalloidin was added to the extracts. Furthermore, three polypeptides of 60 kDa were isolated by binding to DNase I, showing pl values of 6.7, 6.65 and 6.6, less acidic than all reported actin pl values. In A. cylindrica and A. variabilis, specific binding of phalloidin labelled with rhodamine to cells was also detected by spectrofluorometry. In total and soluble cellular extracts, the mAb recognized two bands of 45 and 40 kDa by Western blot analysis, but only the first was purified by binding to DNase I, and it showed three isoforms of pl values 6.8, 6.5 and 6.4. These results suggest the presence, in prokaryotes, of proteins with similar biochemical characteristics to eukaryotic actin.

The metabolism of Clostridium butyricum DSM 5431 was studied in chemostat culture under carbon limitation using either glucose or glycerol. On glycerol, the enzymes glycerol dehydrogenase, diol dehydratase and 1,3-propanediol (1,3-PD) dehydrogenase constitute the branch point that partitions the carbon flux between the competing pathways, i.e. formation of either 1,3-PD or acetate and butyrate. The increasing levels of these enzyme activities with increasing dilution rates (D) explained the constant proportion of glycerol conversion into 1,3-PD. The production of acetate or butyrate constitutes another important branch point and when D increased (i) large amounts of intracellular acetyl-CoA accumulated, (ii) the carbon flux switched from butyric acid to acetic acid, (iii) the specific activity of thiolase was not affected, suggesting this enzyme may be the bottleneck for carbon flux to butyrate biosynthesis providing an explanation for the accumulation of large amounts of intracellular acetyl-CoA, and (iv) high levels of NADH were found in the cell. Oxidation of NADH by 1,3-PD dehydrogenase was linked to the production of 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase. The fact that high intracellular concentrations of NADH were found means that diol dehydratase activity is the rate-limiting step in 1,3-PD formation, avoiding the accumulation of 3-HPA which is a very toxic compound. The specific rate of glucose catabolism (q glucose = 11.1 mmol h-1 g-1) was around four times lower than the specific rate of glycerol catabolism (q glucose = 57.4 mmol h-1 g-1). On glucose-grown cells, reducing equivalents which are released in the glycolytic pathway were reoxidized by the butyric pathway and the low specific formation rate of butyric acid led to an increase in the intracellular level of acetyl-CoA and NADH. Carbon flow was higher on glycerol due to the reoxidation of NADH by both butyric and PD pathways.

The existence of multiple transport systems for Mn2+ in Saccharomyces cerevisiae has been demonstrated in this study. Mn2+ (supplied as MnCI2) was accumulated by S. cerevisiae at all Mn2+ concentrations examined (25 nM-1 mM) but a log-log plot of uptake rates and total amounts accumulated revealed the existence of at least two Mn2+ concentration-dependent transport systems. Over a low Mn2+ concentration range (25-1000 nM), high-affinity Mn2+ uptake occurred with a K m value of 0.3 μM, while transformation of kinetic data obtained over the concentration range 5-200 μM revealed another system with a K m of 62 μM. Meaningful kinetic analyses were not possible at higher Mn2+ concentrations because of toxicity: only about 30% of cells remained viable after 30 min incubation with 1000 μM MnCI2. Release of K+ accompanied Mn2+ accumulation and this increased with increasing Mn2+ concentration. However, even in non-toxic Mn2+ concentrations, the ratio of Mn2+ uptake to K+ release greatly exceeded electroneutral stoichiometric exchange. In 50 μM MnCI2, the ratio was 1: 123 and this increased to 1:2670 in 1000 μM MnCI2, a toxic concentration. External Mg2+ was found to decrease Mn2+ accumulation at all concentrations examined, but to differing extents. Over the low Mn2+ concentration range (5-200 μM), Mg2+ competitively inhibited Mn2+ uptake with a half-maximal inhibitory concentration, K i, of 5.5 μM Mg2+. However, even in the presence of a 50-fold excess of Mg2+, inhibition of Mn2+ uptake was of the order of 72% and it appears that the cellular requirement for Mn2+ could be maintained even in the presence of such a large excess of Mg2+. Over the high Mn2+ concentration range (5-200 μM), the K i for Mg2+ was 25.2 μM. At low Mn2+ concentrations, Zn2+ and Co2+, but not Cd2+, inhibited Mn2+ uptake, which indicated that the high-affinity Mn2+ uptake system was of low specificity, while at higher Mn2+ concentrations, where the lower-affinity Mn2+ transport system operated, inhibition was less marked. However, competition studies with potentially toxic metal cations were complicated due to toxic effects, particularly noticeable at 50 μM Co2+ and Cd2+.

Our understanding of microbial diversity is greatly hampered by the inability to culture as much as 99% of the microbial community in the biosphere. Development of methods for identification and determining microbial phylogenies based on gene sequences, and for recovering genes directly from diverse environmental samples has made it possible to study microbes without the need for cultivation. PCR techniques have revolutionized retrieval of conserved gene sequences. However, it is well known that co-amplification of homologous genes may generate chimeric sequences leading to descriptions of non-existent species. To quantify the frequency of chimeric sequences in PCR amplification of 16S rRNA genes, we chose several 16S rDNAs with known sequences and mixed them for PCR amplifications under various conditions. Using this model system, we detected 30% occurrence of chimeric sequences after 30 cycles of co-amplification of two nearly identical 16S rRNA genes. The frequency of chimera formation decreased to 12.9% and 14.7% for templates with 82% and 86% similarity, respectively. We also examined effects of the number of amplification cycles, length of elongation periods and presence of damaged DNA on chimera formation. The results should provide useful information for microbiologists who use PCR-based strategies to retrieve conserved genes from mixed genomes.

Three 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial isolates were obtained from the highly saline and alkaline Alkali Lake site in southwestern Oregon contaminated with 2,4-D production wastes. While similar in most respects, the three isolates differed significantly in 2,4-D degradation rates, with the most active strain, 1-18, demonstrating an ability to degrade up to 3000 mg 2,4-D I-1 in 3 d. This strain was well adapted to the extreme environment from which it was isolated, growing optimally on 2,4-D at pH 8.4-9.4 and at sodium ion concentrations of 0.6-1.0 M. According to its optimum salt concentration and pH for growth, this isolate was a moderately halophilic, alkaliphilic bacterium. The 16S RNA gene sequence (303 nt) was identical for all three isolates and most closely resembled those of the moderately halophilic eubacteria of the family Halomonadaceae (91% identity). Biochemical and genetic examination revealed strain 1-18 utilizes the same 2,4-D degradation pathway as most of the 2,4-D-degrading bacteria from non-extreme environments. Hybridization data and comparison of the partial sequences of the tfdA gene from the Alkali Lake isolates with those of bacteria from non-extreme environments suggested a common genetic origin of the 2,4-D degradation pathway in the two groups of micro-organisms.

In the course of Streptomyces differentiation, glycogen is accumulated in two discrete phases: in substrate hyphae that undergo aerial mycelium formation (phase I), and during septation of aerial hyphae (phase II). We have disrupted a previously identified gene, glgB, encoding a putative glycogen-branching enzyme in Streptomyces aureofaciens. Disruption of the gene had no profound effect on sporulation. However, the amount of glycogen-like polysaccharides, compared to wild-type (WT) S. aureofaciens, decreased in the late stage of differentiation of the glgB-disrupted strain. Absorption spectra of polysaccharides extracted from the WT and glgB-disrupted strains have shown the presence of glycogen in both strains in the first stage of differentiation (aerial mycelium formation), and unbranched glucan was detected in the glgB-disrupted strain in the late stage of differentiation. The results were confirmed by electron microscopy after silver proteinate staining of glycogen granules. Two distinct glycogen-branching enzymes, which had temporally different expression during differentiation, were detected in WT S. aureofaciens. The absence of this enzyme activity in the late stage of differentiation in the glgB mutant suggests that the product of the glgB gene is responsible for phase II glycogen accumulation.

The P11 protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes. The glnB gene of Rhodospirillum rubrum was isolated and sequenced. The deduced amino acid sequence had very high sequence identity to other P11 proteins. The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced. glnB is cotranscribed with glnA from a promoter with high similarity to the s54-dependent promoter consensus sequence. A putative s70 promoter was also identified further upstream of glnB. Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA. The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.

A procedure to transform intact Lactobacillus sake cells by electroporation was developed through a systematic examination of the effect of changes in various parameters on the transformation efficiency of Lact. sake strain 64F. The most critical factors were found to be the electrical parameters, the composition of washing and electroporation/storage solutions, and the presence of MgCI2 in the expression medium. Under optimal conditions transformation efficiencies up to 107 transformants (μg supercoiled DNA)-1 were obtained. The optimized procedure was successfully applied to other Lact. sake strains and consistently yielded from 104 to 107 transformants (μg supercoiled DNA)-1.

Nisin is a small post-translationally modified lanthionine-containing peptide (lantibiotic) produced by certain Lactococcus lactis strains which has a high antimicrobial activity against several pathogenic Gram-positive bacteria. Northern blots and RT/PCR analyses of the nisin-producing strain N8 revealed that the nisZBTCIPRKFEG gene cluster, responsible for nisin biosynthesis, immunity and regulation, consists of two operons, nisZBTCIPRK and nisFEG. The promoter of the nisFEG operon was mapped. The −35 to −1 region upstream of the transcription start of the nisFEG promoter showed 73% identity with the corresponding region upstream of the nisA and nisZ gene. In contrast to earlier reports, nisin was found to be secreted during the early stages of growth as well as later in the growth cycle. The secreted nisin was adsorbed on the surface of the cells and was released to the medium during mid-exponential growth, when the pH in the medium fell below 5.5. In nisZB antisense and nisT deletion mutant strains constructed in this study the transcription of the nisin operons, nisin production and immunity were lost. Provision of external nisin restored the transcription of both operons in the mutant strains, showing that the operons are coordinately regulated by mature nisin. Nisin induction of the mutant strains also resulted in an increased amount of the Nisl protein and an increase in the level of immunity. Induction using higher concentrations of nisin yielded a higher level of immunity. These results showed that the nisin promoters are under positive control in an autoregulatory manner and that antimicrobial peptides can also function as signal molecules.

Methane is oxidized to methanol by the enzyme methane mono-oxygenase (MMO) in methanotrophic bacteria. In previous work, this multicomponent enzyme system has been extensively characterized at the biochemical and molecular level. Copper ions have been shown to irreversibly inhibit MMO activity in vivo and in vitro, but the effect of copper ions on transcription of the genes encoding the soluble (cytoplasmic) MMO (sMMO) has not previously been investigated. To examine more closely the regulation of bacterial methane oxidation and to determine the role of copper in this process, we have investigated transcriptional regulation of the sMMO gene cluster in the methanotrophic bacterium Methylococcus capsulatus (Bath). Using Northern blot analysis and primer extension experiments, it was shown that the six ORFs of the sMMO gene cluster are organized as an operon and the transcripts produced upon expression of this operon have been identified. The synthesis of these transcripts was under control of a single copper-regulated promoter, which is as yet not precisely defined.

Relatively limited information about promoter structures in Corynebacterium glutamicum has been available until now. With the aim of isolating and characterizing such transcription initiation signals, random Sau3A fragments of C. glutamicum chromosomal DNA and of the corynebacterial phage øGA1 were cloned into the promoter probe vector pEKplCm and selected for promoter activity by chloramphenicol resistance of transformed C. glutamicum cells. The nucleotide sequence of ten chromosomal and three phage fragments was determined and the transcriptional start (TS) sites were localized by primer extension analyses. Additionally, the promoters of five previously isolated C. glutamicum genes were cloned and mapped. All of the isolated promoters were also functional in the heterologous host Escherichia coli. A comparative analysis of the newly characterized promoter sequences together with published promoters from C. glutamicum revealed conserved sequences centred about 35 bp (ttGcca) and 10 bp (TA.aaT) upstream of the TS site. The position of these motifs and the motifs themselves are comparable to the −35 and −10 promoter consensus sequences of other Gram-positive and Gram-negative bacteria, indicating that they represent transcription initiation signals in C. glutamicum. However, the C. glutamicum consensus hexamer of the −35 region is much less conserved than in E. coli, Bacillus, Lactobacillus and Streptococcus.

A transcriptional regulator gene, designated adiY, was found downstream of the biodegradative arginine decarboxylase (adiA) gene (previously known as adi) of Escherichia coli. The arginine decarboxylase system is maximally induced under conditions of acidic pH, anaerobiosis and rich medium, and AdiY was found to increase the expression of adiA. The DNA sequence of adiY encodes a protein of 253 amino acids. Primer extension analysis defined the promoter. The amino acid sequence of AdiY showed homology to the XyIS/AraC family of transcriptional regulators, which includes EnvY and AppY. Studies suggested that sequences required for acid induction were also necessary to observe the stimulation by AdiY. An examination of the substitution of AdiY, AppY and EnvY showed that these three proteins can, to some extent, stimulate the other systems.

The SDS-insoluble protein fraction of Agaricus bisporus fruiting bodies was solubilized with trifluoroacetic acid. On SDS-PAGE this fraction was found to contain one abundant protein with an apparent M r of 16 kDa. The N-terminal amino acid sequence of this protein was determined and RT-PCR used to isolate a cDNA clone which upon sequencing identified the protein as a typical class I hydrophobin (ABH1). The gene (ABH1) was isolated and sequenced, and a second hydrophobin gene (ABH2) was found about 2.5 kbp downstream of ABH1. Purified ABH1 self-assembled at hydrophobic-hydrophilic interfaces, producing the typical rodlet layer known from other hydrophobins. Similar rodlets were observed on the surface of the fruiting body, while immunological localization showed the hydrophobin to be particularly abundant at the outer surface of fruiting bodies, in the veil and in the core tissue of the stipe. Transcripts of ABH1 were found only in fruiting-body hyphae. The ABH1 hydrophobin is probably solely responsible for the hydrophobicity of the fruiting-body surface but may also line air channels within fruiting bodies.