Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria

A phylogenetic tree constructed by 5S rRNA analysis is composed of three major branches in eubacteria: high G + C Gram+, low G + C Gram+ and Gram- [Hori and Osawa, Mol. Biol. Evol. 4 (1987) 445-472]. We have shown that the characteristic dnaA region is common among Escherichia coli (Gram-), Pseudomonas putida (Gram-), and Bacillus subtilis (low G + C Gram+). We have now determined the structure of the dnaA region of Micrococcus luteus, as a representative of the last branch, high G + C Gram+. The dnaA gene and at least three other genes, rnpA, rpmH and dnaN were found to be conserved in M. luteus. Large nontranslatable regions were found flanking the dnaA gene. The upstream region is conserved in the four bacteria so far examined. On the other hand, the downstream region is conserved only in Gram+ bacteria, M. luteus and B. subtilis. The consensus sequence of the DnaA box in M. luteus seems to be TTGTCCACA, in contrast to TTATCCACA of other bacteria. These results confirm our hypothesis that the dnaA region is the replication origin of the ancestral bacteria and that the essential feature of the DnaA protein and DnaA-box combination is conserved in eubacteria.     

Cloning and sequencing of the gene encoding glutamine synthetase I from the archaeum Pyrococcus woesei: anomalous phylogenies inferred from analysis of archaeal and bacterial glutamine synthetase I sequences.

The gene glnA encoding glutamine synthetase I (GSI) from the archaeum Pyrococcus woesei was cloned and sequenced with the Sulfolobus solfataricus glnA gene as the probe. An operon reading frame of 448 amino acids was identified within a DNA segment of 1,528 bp. The encoded protein was 49% identical with the GSI of Methanococcus voltae and exhibited conserved regions characteristic of the GSI family. The P. woesei GSI was aligned with available homologs from other archaea (S. solfataricus, M. voltae) and with representative sequences from cyanobacteria, proteobacteria, and gram-positive bacteria. Phylogenetic trees were constructed from both the amino acid and the nucleotide sequence alignments. In accordance with the sequence similarities, archaeal and bacterial sequences did not segregate on a phylogeny. On the basis of sequence signatures, the GSI trees could be subdivided into two ensembles. One encompassed the GSI of cyanobacteria and proteobacteria, but also that of the high-G + C gram-positive bacterium Streptomyces coelicolor (all of which are regulated by the reversible adenylylation of the enzyme subunits); the other embraced the GSI of the three archaea as well as that of the low-G + C gram-positive bacteria (Clostridium acetobutilycum, Bacillus subtilis) and Thermotoga maritima (none of which are regulated by subunit adenylylation). The GSIs of the Thermotoga and the Bacillus-Clostridium lineages shared a direct common ancestor with that of P. woesei and the methanogens and were unrelated to their homologs from cyanobacteria, proteobacteria, and S. coelicolor. The possibility is presented that the GSI gene arose among the archaea and was then laterally transferred from some early methanogen to a Thermotoga-like organism. However, the relationship of the cyanobacterial-proteobacterial GSIs to the Thermotoga GSI and the GSI of low-G+C gram-positive bacteria remains unexplained.     

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.     

Role of GC-biased mutation pressure on synonymous codon choice in Micrococcus luteus, a bacterium with a high genomic GC-content.

The GC (G + C, or G or C)-contents of codon silent positions in all two-codon sets and three codons AUY/A (IIe), and in most of the family boxes of Micrococcus luteus (genomic GC-content: 74%) are 95% to 100% in both the highly and weakly expressed genes. In some family boxes, there is a decrease in NNC codons and an increase in NNG codons from the highly expressed to weakly expressed genes without apparent involvement of NNU and NNA codons. From these observations, we conclude that the selective use of synonymous codons in M. luteus may be largely determined by GC-biased mutation pressure and that in the highly expressed genes tRNAs would act as a weak selection pressure in some family boxes. Available data suggest that the effect of selection pressure by tRNAs on the synonymous codon choice becomes more apparent in the highly expressed genes in eubacteria with intermediate GC-contents such as Escherichia coli and Bacillus subtilis, and that the U/C ratio of the codon third positions in NNU/C-type two-codon sets in the weakly expressed genes would represent the approximate magnitude of directional mutation pressure throughout eubacteria.     

Secondary structure and phylogeny of Staphylococcus and Micrococcus 5S rRNAs

Nucleotide sequences of 5S rRNAs from four bacteria, Staphylococcus aureus Smith (diffuse), Staphylococcus epidermidis ATCC 14990, Micrococcus luteus ATCC 9341 and Micrococcus luteus ATCC 4698, were determined. The secondary structural models of S. aureus and S. epidermidis sequences showed characteristics of the gram-positive bacterial 5S rRNA (116-N type [H. Hori and S. Osawa, Proc. Natl. Acad. Sci. U.S.A. 76:381-385, 1979]). Those of M. luteus ATCC 9341 and M. luteus ATCC 4698 together with that of Streptomyces griseus (A. Simoncsits, Nucleic Acids Res. 8:4111-4124, 1980) showed intermediary characteristics between the gram-positive and gram-negative (120-N type [H. Hori and S. Osawa, 1979]) 5S rRNAs. This and previous studies revealed that there exist at least three major groups of eubacteria having distinct 5S rRNA and belonging to different stems in the 5S rRNA phylogenic tree.     

A phylogenetic analysis of micro-organisms isolated from subsurface environments

Three methods were used to provide information on the identity and phylogenetic relatedness of 19 aerobic, chemoheterotrophic bacteria isolated from topsoil and deep subsurface sediments at a site in South Carolina. These methods were (i) analysis of selected physiological traits, (ii) restriction endonuclease analysis (REA) of genomic DNA, and (iii) analysis of 16S ribosomal RNA sequences. When the 16S rRNA sequences were compared with those for 12 standard strains, two topsoil isolates and six subsurface strains formed a tight group with the high-G+C Gram-positive bacteria and appeared to be most closely related to Arthrobacter globiformis— a coryneform-actinomycete bacterium with unusually effective survival capabilities. The rest of the subsurface isolates were scattered among the standard strains from the Proteobacteria— including the pseudomonads and Agrobacterium tumefaciens— or the low-G+C Gram-positive bacteria.     

Prophage genomics.

The majority of the bacterial genome sequences deposited in the National Center for Biotechnology Information database contain prophage sequences. Analysis of the prophages suggested that after being integrated into bacterial genomes, they undergo a complex decay process consisting of inactivating point mutations, genome rearrangements, modular exchanges, invasion by further mobile DNA elements, and massive DNA deletion. We review the technical difficulties in defining such altered prophage sequences in bacterial genomes and discuss theoretical frameworks for the phage-bacterium interaction at the genomic level. The published genome sequences from three groups of eubacteria (low- and high-G+C gram-positive bacteria and gamma-proteobacteria) were screened for prophage sequences. The prophages from Streptococcus pyogenes served as test case for theoretical predictions of the role of prophages in the evolution of pathogenic bacteria. The genomes from further human, animal, and plant pathogens, as well as commensal and free-living bacteria, were included in the analysis to see whether the same principles of prophage genomics apply for bacteria living in different ecological niches and coming from distinct phylogenetical affinities. The effect of selection pressure on the host bacterium is apparently an important force shaping the prophage genomes in low-G+C gram-positive bacteria and gamma-proteobacteria.     

Changes in the fine structure of microbial cells induced by chaotropic salts

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

DNA Polymerase C of the Thermophilic Bacterium Thermus aquaticus: Classification and Phylogenetic Analysis of the Family C DNA Polymerases

Bacterial family C DNA polymerases (DNA pol IIIs), the major chromosomal replicative enzymes, have been provisionally classified based on primary sequences and domain structures into three classes: class I (Escherichia coli DNA pol C-type), class II (Bacillus subtilis DNA pol C-type), and class III (cyanobacterial DNA pol C-type), respectively. We have sequenced the structural gene encoding the DNA pol C catalytic subunit of the thermophilic bacterium Thermus aquaticus. This gene, designated the Taq DNA pol C gene, contains a 3660-bp open reading frame which specifies a polypeptide of molecular weight of 137,388 daltons. Comparative sequence analyses revealed that Taq DNA pol C is a class I family C DNA polymerase. The Taq DNA pol C is most closely related to the Deinococcus radiodurans DNA pol C. Although a phylogenetic tree based on the class I family C DNA pols is still in the provisional stage, some important conclusion can be drawn. First, the high-G+C and the low-G+C Gram-positive bacteria are not monophyletic. Second, the low-G+C Gram-positive bacteria contain multigenes of family C DNA pols (classes I and II). Third, the cyanobacterial family C DNA pol, classified as class III because it is encoded by a split gene, forms a group with the high-G+C Gram-positive bacteria. Received: 7 October 1998 / Accepted: 12 January 1999     

Menaquinol oxidase activity and primary structure of cytochrome bd from the amino-acid fermenting bacterium Corynebacterium glutamicum

Cytochrome d was spectroscopically detected in membrane fractions of the amino-acid-fermenting, high-G+C gram-positive bacterium Corynebacterium glutamicum. Inhibition of NADH oxidase activity in the membranes by cyanide suggested that the main terminal respiratory oxidase during the stationary phase was a type of cytochrome bd. Cytochrome bd-type quinol oxidase, purified from the membranes, was composed of two subunits. Its reduced form showed absorption peaks at 627, 595, and 560 nm, which were due to haem d, high-spin protohaem, and low-spin protohaem, respectively. The air-oxidised form showed a peak at 645 nm, which might be due to oxygenated ferrous haem d. The spectral features and the size of subunit I are more similar to the properties of cytochromes bd from Proteobacteria, such as Escherichia coli, than to those of cytochrome bd from low-G+C gram-positive bacteria, such as Bacillus stearothermophilus. The menaquinol oxidase activity of the purified cytochrome bd was low, but was enhanced about fivefold by pre-incubating the enzyme with menaquinones. The order of effectiveness of quinols as oxidase substrates was clearly different from that of quinones as the activators of enzyme activity. Furthermore, activation was destroyed by ultraviolet irradiation of the pre-incubated enzyme and then restored by a second incubation with menaquinone. These results indicate that the enzymatic properties of this new oxidase are more similar to the properties of cytochromes bd from low-G+C gram-positive bacterial than to those of proteobacterial counterparts. They also suggest that the enzyme has a second quinone-binding site essential for full activity, in addition to the active centre for substrate oxidation. By using probes based on partial peptide sequences of the subunits, the genes for the two subunits of C. glutamicum cytochrome bd were cloned. The deduced amino acid sequence demonstrated that subunit I lacks the C-terminal half of the Q loop and that the primary structure of C. glutamicum cytochrome bd is more similar to that of other gram-positive bacteria than to proteobacterial cytochromes bd.     

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.     

Study of ectoparasitism of ultramicrobacteria of the genus Kaistia, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gram-negative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated, but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

16S rRNA sequences and differences in bacteria isolated from the Muztag Ata glacier at increasing depths

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2 degrees C or -2 degrees C up to approximately 20 degrees C), although the majority (82%) were psychrotolerant (grew at 2 degrees C or -2 degrees C up to 37 degrees C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were gamma-Proteobacteria, 14.7% were alpha-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.     

Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 microm3) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5 degrees C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-microm, 0.2-microm, and even 0.1-microm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-microm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.     

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.     

Molecular systematic studies of eubacteria, using sigma70-type sigma factors of group 1 and group 2.

Sigma factors of the sigma70 family were used as a phylogenetic tool to compare evolutionary relationships among eubacteria. Several new sigma factor genes were cloned and sequenced to increase the variety of available sequences. Forty-two group 1 sigma factor sequences of various species were analyzed with the help of a distance matrix method to establish a phylogenetic tree. The tree derived by using sigma factors yielded subdivisions, including low-G+C and high-G+C gram-positive bacteria, cyanobacteria, and the alpha, beta, gamma, and delta subdivisions of proteobacteria, consistent with major bacterial groups found in trees derived from analyses with other molecules. However, some groupings (e.g., the chlamydiae, mycoplasmas, and green sulfur bacteria) are found in different positions than for trees obtained by using other molecular markers. A direct comparison to the most extensively used molecule in systematic studies, small-subunit rRNA, was made by deriving trees from essentially the same species set and using similar phylogenetic methods. Differences and similarities based on the two markers are discussed. Additionally, 31 group 2 sigma factors were analyzed in combination with the group 1 proteins in order to detect functional groupings of these alternative sigma factors. The data suggest that promoters recognized by the major vegetative sigma factors of eubacteria will contain sequence motifs and spacing very similar to those for the sigma70 sigma factors of Escherichia coli.     

ATP and polyphosphate-dependent bacterial NAD+-kinases

Measurable levels of activity of NAD+ kinases of actinomycetes Micrococcus luteus and Corynebacterium ammoniagenes were observed after substituting inorganic tripolyphosphate for ATP, whereas the enzyme from the eubacterium Escherichia coli was not active with this substrate. Gradient PAGE found two molecular isoforms of NAD+ kinase in C. ammoniagenes and E. coli; four forms were found in M. luteus. All isoforms of this enzyme found in C. ammoniagenes and M. luteus displayed a NADP-synthesizing activity in the presence of either ATP or tripolyphosphate. Because of its capability of utilizing inorganic tripolyphosphate, M. luteus is the most promising NADP producer organism.     

Depth distribution of microbial diversity in Mono Lake,a meromictic soda lake in California

We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain BACTERIA: alpha- and gamma-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented beta- and delta-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.