Ribsome and messenger specificity in protein synthesis by bacteria

Ribosomes from Escherichia coli and Pseudomonas fluorescens, two Gram-negative bacteria, translated messenger RNA preparations from all bacteria tested (3 Gram-negative species, and 6 Gram-positive species) as well as f2 RNA and T4 early messenger RNA. Ribosomes from Clostridium pasteurianum, Streptococcus faecalis, and Bacillus subtilis, three Gram-positive organisms, did not translate messenger RNA preparations derived from any of the Gram-negative organisms, the f2 RNA, or the T4 early messenger RNA but did translate messenger preparations from all 6 Gram-positive bacterial species tested.     

Biodiversity of moderately halophilic bacteria in hypersaline habitats in Egypt

Screening bacteria from different saline environments in Alexandria. Egypt, lead to the isolation of 76 Gram-negative and 14 Gram-positive moderately halophilic bacteria. The isolates were characterized taxonomically for a total of 155 features. These results were analyzed by numerical techniques using simple matching coefficient (SSM) and the clustering was achieved by the unweighed pair-group method of association (UPGMA). At 75% similarity level the Gram-negative bacteria were clustered in 7 phena in addition to one single isolate, whereas 4 phena represented the Gram-positive. Based on phenotypic characteristics, it is suggested that the Gram-negative bacteria belong to the genera Pseudoalteromonas, Flavobacterium, Chromohalobacter, Halomonas and Salegentibacter, in addition to the non-identified single isolate. The Gram-positive bacteria are proposed to belong to the genera Halobacillus, Salinicoccus, Staphylococcus and Tetragenococcus. This study provides the first publication on the biodiversity of moderately halophilic bacteria in saline environments in Alexandria, Egypt.     

Comparison of ribosomes from gram-positive and gram-negative bacteria with respect to the presence of protein S1

Ribosomes from Gram-positive and Gram-negative bacteria have been analysed for the presence of ribosomal protein S1 by three methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoreaction with E. coli anti-S1 and chromatography on poly (U)-Sepharose. We observed that protein S1 is predominantly present in Gram-negative bacteria in comparison with Gram-positive bacteria. Exceptions are noted in both species.     

Diversity analysis of diazotrophic bacteria associated with the roots of tea (Camellia sinensis (L.) O. Kuntze)

The diversity elucidation by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of 96 associative diazotrophs, isolated from the feeder roots of tea on enriched nitrogen-free semisolid media, revealed the predominance of Gram-positive over Gram-negative bacteria within the Kangra valley in Himachal Pradesh, India. The Gram-positive bacteria observed belong to two taxonomic groupings; Firmicutes, including the genera Bacillus and Paenibacillus; and Actinobacteria, represented by the genus Microbacterium. The Gram-negative bacteria included alpha-Proteobacteria genera Brevundimonas, Rhizobium, and Mesorhizobium; gamma-Proteobacteria genera Pseudomonas and Stenotrophomonas; and beta-Proteobacteria genera Azospira, Burkholderia, Delftia, Herbaspirillum and Ralstonia. The low level of similarity of two isolates, with the type strains Paenibacillus xinjiangensis and Mesorhizobium albiziae, suggests the possibility of raising species novum. The bacterial strains of different phylogenetic groups exhibited distinct carbon-source utilization patterns and fatty acid methyl ester profiles. The strains differed in their nitrogenase activities with relatively high activity seen in the Gramnegative strains exhibiting the highest similarity to Azospira oryzae, Delftia lacustris and Herbaspirillum huttiense.     

Rapid identification of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> by species-specific primers based on the genes involved in the Entner–Doudoroff pathway

In this study we report a novel method for identification of Enterococcus faecalis based on polymerase chain reaction with primers specific for the eda-genes encoding the enzymes involved in the Entner-Doudoroff pathway, a pathway present only in this species among Gram-positive bacteria. The designed primers were checked in several different Enterococcus species, and with some other Gram-positive and Gram-negative bacterial species as well. Five primer combinations were used to detect the eda-1 gene, and another three for the eda-2 gene. With the exception of one of the primer combinations, all the others gave as results the expected amplification products only in E. faecalis strains.     

16S rRNA analysis of Sporomusa,selenomonas, and Megasphaera: on the phylogenetic origin of Gram-positive Eubacteria

In order to determine the phylogenetic relationships of representatives of three Gram-negative genera, Sporomusa, Selenomonas, and Megasphaera, the 16S ribosomal RNAs were compared by oligonucleotide cataloguing. Surprisingly, Sporomusa ovata, S. sphaeroides, Selenomonas ruminantium, and Megasphaera elsdenii do not group with any of the 200 Gram-negative eubacterial species investigated so far by this method but show a distinct although remote relationship to Gram-positive eubacteria of the Clostridium subdivision. The presence of Gram-negative species within the radiation of the cluster defined by Gram-positive cubacteria reduces the significance the Gram-positive staining behaviour plays in taxonomic and phylogenetic studies. It furthermore supports previous findings showing the Gram-negative and phototrophic species Heliobacterium chlorum to be a member of the Clostridium-Bacillus cluster. The presence of Gram-negative endospore-containing Sporomusa species among the 16S rRNA cluster of Gram-positive endospore-forming eubacteria allow speculations about the evolutionary origin of Gram-positive eubacteria.This paper is dedicated to Professor Dr. O. Kandler, on the occasion of his 65th birthdayEs was supported by the Gesellschaft für Biotechnologische Forschung (GBF) for performing research of relevance for the German Collection of Microorganisms (DSM). CRW was supported by a grant, DEB-8107061, from the National Science Foundation     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Identification of cyanobacterial cell division genes by comparative and mutational analyses

We performed comparative and mutational analyses to define more comprehensively the repertoire of genes involved in cyanobacterial cell division. Genes ftsE, ftsI, ftsQ, ftsW, and (previously recognized) ftsZ, minC, minD, minE and sulA were identified as homologues of cell division genes of Gram-negative and Gram-positive bacteria. Transposon mutagenesis of Synechococcus elongatus PCC 7942 identified five additional genes, cdv1, cdv2, cdv3, ftn6 and cikA, involved in cell division. cdv1 encodes a presumptive periplasmic peptidyl-prolyl cis-trans isomerase. cdv2 has similarity to ylmF which, like divIVA, lies within the Gram-positive bacterial ylm gene cluster whose members have functions associated with division. Conservation of other ylm genes in cyanobacteria suggests that cyanobacteria and Gram-positive bacteria share specific division proteins. Two ylm homologues are also found in algal and plant genomes. cdv3 has low but significant similarity to divIVA, suggesting that minE and cdv3 both mediate division-site determination in cyanobacteria. In contrast, Gram-positive bacteria lack minE, and (Gram-negative) proteobacteria lack divIVA. ftn6, of unknown function, and the circadian input kinase, cikA, are specific to cyanobacteria. In S. elongatus, unlike in other bacteria, FtsZ rings are formed at sites occupied by nucleoids. Thus, the division machinery of cyanobacteria differs in its composition and regulation from that of Gram-negative and Gram-positive bacteria.     

三种百合鳞茎提取物的抑菌作用

采用纸片琼脂扩散法测定了宜昌百合、岷江百合及兰州百合鳞茎甲醇提取物对革兰氏阳性细菌(金黄色葡萄球菌、枯草芽孢杆菌)和革兰氏阴性细菌(大肠杆菌、沙门氏菌)的抑制活性,并对3种百合鳞茎提取物的含量与抑菌活性进行了剂量-效应关系分析。结果表明:3种百合鳞茎提取物对4种细菌均具有抑制活性,对革兰氏阳性细菌的抑菌活性高于对革兰氏阴性细菌的抑菌活性,且宜昌百合和岷江百合两种野生百合鳞茎提取物的抑菌活性均高于普通食用的兰州百合;3种百合鳞茎提取物的含量与抑菌活性之间存在明显的剂量-效应关系,即随着提取物含量的升高,抑菌活性明显升高。     

Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.

Invasive infection with Gram-positive and Gram-negative bacteria often results in septic shock and death. The basis for the earliest steps in innate immune response to Gram-positive bacterial infection is poorly understood. The LPS component of the Gram-negative bacterial cell wall appears to activate cells via CD14 and Toll-like receptor (TLR) 2 and TLR4. We hypothesized that Gram-positive bacteria might also be recognized by TLRs. Heterologous expression of human TLR2, but not TLR4, in fibroblasts conferred responsiveness to Staphylococcus aureus and Streptococcus pneumoniae as evidenced by inducible translocation of NF-kappaB. CD14 coexpression synergistically enhanced TLR2-mediated activation. To determine which components of Gram-positive cell walls activate Toll proteins, we tested a soluble preparation of peptidoglycan prepared from S. aureus. Soluble peptidoglycan substituted for whole organisms. These data suggest that the similarity of clinical response to invasive infection by Gram-positive and Gram-negative bacteria is due to bacterial recognition via similar TLRs.     

Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in Gram-positive bacteria

A 1.5 kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the 'rolling-circle' mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the 'rolling-circle' group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.     

IS257 from Staphylococcus aureus: member of an insertion sequence superfamily prevalent among gram-positive and gram-negative bacteria

The nucleotide sequences for the IS257 family of insertion sequences from Staphylococcus aureus were compared with those of the ISS1 family from Streptococcus lactis and the IS15 family which is widespread amongst Gram-negative bacteria. These elements have a striking degree of similarity in both their putative transposase polypeptide sequences and their nucleotide sequences (40 to 64% between pairs), including 12 out of 14 bp conservation in their terminal inverted repeats. The evolutionary distance between the IS15 family and the IS257 and ISS1 families of Gram-positive origin is approximately twice that between the IS257 and ISS1 families. Analysis of base substitutions in the three sequences has provided insights into the effect of selection for the G + C content of immigrant genes to conform to that of their hosts, and into the evolution of biases in overall amino acid composition of cellular proteins in prokaryotes and eukaryotes. The IS257, ISS1, IS15 families form a superfamily of insertion sequences that has been involved in the spread of a number of antimicrobial resistance determinants in Gram-positive and Gram-negative pathogens.     

Interaction of the calcium ionophore A.23187 (Calcimycin) with Bacillus cereus and Escherichia coli

Ionophores isolated from bacterial strains, and especially A.23187, are efficient antibiotics against Gram-positive bacteria and devoid of activity on Gram-negative species. This difference in activity was attributed to the outer membrane of Gramnegative bacteria which is presumably impermeable to these very hydrophobic compounds. In this context, the partition of the calcium ionophore A.23187 between bacteria and the medium was studied on Escherichia coli (Gram-negative) and Bacillus cereus (Gram-positive) using, on the one hand, fluorimetric measurements and, on the other hand, radioautographic analysis of bacteria incubated with the [³H]-labelled ionophore. Although the first method did not give a definitive answer, the second one clearly showed that the tritiated metabolite was only incorporated into B. cereus.     

Translocation of proteins across the cell envelope of Gram-positive bacteria

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.     

Secretion, processing and activation of bacterial extracellular proteases

Many different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram-positive bacteria are secreted by a signal-peptide-dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram-negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram-negative bacteria secrete one or more proteases via a novel signal-peptide-independent route. Most proteases secreted by Gram-negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best-characterized extracellular proteases produced by Gram-positive and Gram-negative bacteria.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus

Background: The evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. The current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved from an archaebacterial ancestor. Recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, particularly regarding the origin of eukaryotic cells.Results We have used sequences of 70 kD heat shock protein (hsp70) — the most conserved protein found to date in all species — to examine the evolutionary relationship between various species. We have obtained two new archaebacterial hsp70 sequences from the species, Thermoplasma acidophilum and Halobacterium cutirubrum. A global comparison of hsp70 sequences, including our two new sequences, shows that all known archaebacterial homologs share a number of sequence signatures with the Gram-positive group of bacteria that are not found in any other prokaryotic or eukaryotic species. In contrast, the eukaryotic homologs are shown to share a number of unique sequence features with the Gram-negative bacteria that are not present in any archaebacteria. Detailed phylogenetic analyses of hsp70 sequences strongly support a specific evolutionary relationship between archaebacteria and Gram-positive bacteria on the one hand, and Gram-negative bacteria and eukaryotes on the other. The phylogenetic analyses also indicate a polyphyletic branching of archaebacteria within the Gram-positive species. The possibility that the observed relationships are due to horizontal gene transfers can be excluded on the basis of sequence characteristics of different groups of homologs.Conclusion Our results do not support the view that archaebacteria constitute a monophyletic domain, but instead suggest a close evolutionary linkage between archaebacteria and Gram-positive bacteria. Furthermore, in contrast to the presently accepted view, eukaryotic hsp70s show a close and specific relationship to those from Gram-negative species. To explain the phylogenies based on different gene sequences, a chimeric model for the origin of the eukaryotic cell nucleus involving fusion between an archaebacterium and a Gram-negative eubacterium is proposed. Several predictions from the chimeric model are discussed.     

Aerobic salivary bacteria in wild and captive Komodo dragons

During the months of November 1996, August 1997, and March 1998, saliva and plasma samples were collected for isolation of aerobic bacteria from 26 wild and 13 captive Komodo dragons (Varanus komodoensis). Twenty-eight Gram-negative and 29 Gram-positive species of bacteria were isolated from the saliva of the 39 Komodo dragons. A greater number of wild than captive dragons were positive for both Gram-negative and Gram-positive bacteria. The average number of bacterial species within the saliva of wild dragons was 46% greater than for captive dragons. While Escherichia coli was the most common bacterium isolated from the saliva of wild dragons, this species was not present in captive dragons. The most common bacteria isolated from the saliva of captive dragons were Staphylococcus capitis and Staphylococcus capitis and Staphylococcus caseolyticus, neither of which were found in wild dragons. High mortality was seen among mice injected with saliva from wild dragons and the only bacterium isolated from the blood of dying mice was Pasteurella multocida. A competitive inhibition enzyme-linked immunosorbent assay revealed the presence of anti-Pasteurella antibody in the plasma of Komodo dragons. Four species of bacteria isolated from dragon saliva showed resistance to one or more of 16 antimicrobics tested. The wide variety of bacteria demonstrated in the saliva of the Komodo dragon in this study, at least one species of which was highly lethal in mice and 54 species of which are known pathogens, support the observation that wounds inflicted by this animal are often associated with sepsis and subsequent bacteremia in prey animals.     

Taxonomy and chemical characterization of new antibiotics produced by Saccharothrix SA198 isolated from a Saharan soil

Actinomycete strain SA198, isolated from a Saharan soil sample of Algeria, exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, and phytopathogenic and toxinogenic fungi. The morphological and chemotaxonomic characteristics of the strain were consistent with those of the genus Saccharothrix. Analysis of the 16S rRNA gene sequence of strain SA198 showed a similarity level ranging between 97.2 and 98.8% within Saccharothrix species, S. australiensis being the most closely related. Two new active products were isolated by reverse HPLC using a C18 column. The ultraviolet–visible (UV–VIS), infrared (IR), mass, and ¹H and ¹⁴C nuclear magnetic resonance (NMR) spectra showed that these products were new bioactive compounds. The minimum inhibitory concentrations of these antibiotics showed a strong activity against fungi and moderate activities against Gram-positive and Gram-negative bacteria.     

Multidrug resistance in hydrocarbon-tolerant Gram-positive and Gram-negative bacteria

New Gram-positive and Gram-negative bacteria were isolated from Poeni oily sludge, using enrichment procedures. The six Gram-positive strains belong to Bacillus, Lysinibacillus and Rhodococcus genera. The eight Gram-negative strains belong to Shewanella, Aeromonas, Pseudomonas and Klebsiella genera. Isolated bacterial strains were tolerant to saturated (i.e., n-hexane, n-heptane, n-decane, n-pentadecane, n-hexadecane, cyclohexane), monoaromatic (i.e., benzene, toluene, styrene, xylene isomers, ethylbenzene, propylbenzene) and polyaromatic (i.e., naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons, and also resistant to different antimicrobial agents (i.e., ampicillin, kanamycin, rhodamine 6G, crystal violet, malachite green, sodium dodecyl sulfate). The presence of hydrophilic antibiotics like ampicillin or kanamycin in liquid LB-Mg medium has no effects on Gram-positive and Gram-negative bacteria resistance to toxic compounds. The results indicated that Gram-negative bacteria are less sensitive to toxic compounds than Gram-positive bacteria, except one bacteria belonging to Lysinibacillus genus. There were observed cellular and molecular modifications induced by ampicillin or kanamycin to isolated bacterial strains. Gram-negative bacteria possessed between two and four catabolic genes (alkB, alkM, alkB/alkB1, todC1, xylM, PAH dioxygenase, catechol 2,3-dioxygenase), compared with Gram-positive bacteria (except one bacteria belonging to Bacillus genus) which possessed one catabolic gene (alkB/alkB1). Transporter genes (HAE1, acrAB) were detected only in Gram-negative bacteria.