Biomedical subjects

R P Mellado

Publications and source records attributed to R P Mellado.

At least 19 recordsLinked to original sources

PubMed2003

Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

Bacillus subtilis↗

PubMed2001

Functional analysis of the Streptomyces lividans type I signal peptidases.

Type I signal peptidases are responsible for the proteolytic cleavage of the signal peptide of secreted proteins. In the gram-positive bacterium Streptomyces lividans, four adjacent genes (sipW, sipX, sipY and sipZ) were isolated encoding putative type I signal peptidases. In this work, the different sip genes were cloned and expressed. Subsequently, the Sip proteins were purified to raise antibodies. Although the four Sip proteins share a low degree of sequence similarity and differ significantly in size and pI, anti-Sip antibodies cross-reacted intensively. Functional signal peptidase processing activity for each of these Sip proteins was shown both in vitro and in vivo. The different Sip proteins did not exhibit the same cleavage efficiency on the Bacillus subtilis pre-chitosanase.

Antibodies, Bacterial↗

PubMed2001

Membrane topology of the Streptomyces lividans type I signal peptidases.

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.

Base Sequence↗

PubMed1999

Streptomyces lividans as a host for the production and secretion of Escherichia coli TEM beta-lactamase.

The regulatory region and the region coding for the signal peptide of an extracellular agarase have been used to synthesize and secrete the heterologous Escherichia coli TEM beta-lactamase in Streptomyces lividans. The transcriptional regulation of the chimeric gene, and the secretion pattern of the chimeric gene product, coincided with those of the agarase gene. The negative glucose effect on the secretion of the protein was reverted when the recombinant bacterium was grown in the chemostat under phosphate limiting conditions.

Culture Media↗

PubMed1998

Metal accumulation and vanadium-induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae.

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family Enterobacteriaceae were selected. Two of them were Escherichia hermannii strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an Enterobacter cloacae strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in E. hermannii and E. cloacae. Incubation with this metal changed the OMP profile of E. hermannii but did not produce variations in the expression of the major OMPs of E. cloacae.

Anti-Bacterial Agents↗

PubMed1998

A new signal peptidase gene from Streptomyces lividans TK21.

Using synthetic oligonucleotides derived from known signal peptidase genes and a multicopy plasmid as a vector, a signal peptidase gene (sipZ) from Streptomyces lividansTK21 has been cloned. The primary structure of the gene has been determined and the amino acid composition of the SipZ protein inferred. SipZ is 258 aa long and showed homology to other type I signal peptidases, containing like them an N-terminal transmembrane anchor. Alignment of SipZ with other known SPases allowed the identification of a conserved sequence of amino acids specific for Gram-positive bacteria.

Amino Acid Sequence↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Overproduction and purification of an agarase of bacterial origin.

The agarase gene from Streptomyces coelicolor has been cloned in the non-producer bacterium Streptomyces lividans under the control of its own set of promoters and under the control of a heterologous promoter that is functional only during exponential growth. The best level of overproduction was obtained when the strain containing the natural gene was cultivated in fed batch with mannitol as carbon source. The protein, with a relative molecular mass of 32 kDa, has been purified following an affinity purification method. Contaminating activities seem to be absent from the purified enzyme preparation that can be used to purify DNA from agarose gels.

Bacterial Proteins↗

PubMed1997

Ribosomal RNA synthesis in Streptomyces lividans under heat shock conditions.

Clones containing rRNA genes were isolated from a gene library of Streptomyces lividans when RNA produced under heat shock conditions was used as a probe. Two of the clones carried entire rRNA operons rrnA and rrnF, respectively, the expression of both operons being under the control of four different promoters. At least two of the promoters were fully functional when the temperature increased from 30 to 45 degrees C, ensuring transcription of the rRNA genes under the heat shock. A third clone carried a partial rRNA operon in which expression was controlled by a main promoter that was functional at both 30 and 45 degrees C.

Base Sequence↗

PubMed1996

A relA/spoT homologous gene from Streptomyces coelicolor A3(2) controls antibiotic biosynthetic genes.

A 0.972-kilobase pair DNA fragment from Streptomyces lividans that induces the production of the blue-pigmented antibiotic actinorhodine in S. lividans when cloned on a multicopy plasmid has led to the isolation of a 4-kilobase pair DNA fragment from Streptomyces coelicolor containing homologous sequence. Computer-assisted analysis of the DNA sequence revealed three putative open reading frames (ORFs), ORF1, ORF2, and ORF3. ORF2 extends beyond the sequenced DNA fragment, and its deduced product shares no similarities with any other known proteins in the data bases. ORF3 is also truncated, and its 41-amino acid C-terminal product is identical to the S. coelicolor adenine phosphoribosyltransferase. The 847-amino acid ORF1 protein, with a predicted molecular mass of 94.2 kDa, strongly resembled the relA and spoT gene products from Escherichia coli and the homologs from Vibrio sp. strain S14, Haemophilus influenzae, Streptococcus equisimilis H46A, and Mycoplasma genitalium. Unlike these proteins, the ORF1 amino acid sequence analysis revealed the presence of a putative ATP/GTP-binding domain. A mutant was generated by deleting most of the ORF1 gene that showed an actinorhodine-nonproducing phenotype, while undecylprodigiosin and the calcium-dependent antibiotic were unaffected. The mutant strain grew at a much lower rate than the wild-type strain, and spore formation was delayed. When the gene was propagated on a low copy number vector, not only was actinorhodine production restored, but actinorhodine and undecylprodigiosin production was enhanced in both the mutant and wild-type and morphological differentiation returned to wild-type characteristics. (p)ppGpp synthetase activity was not detected in purified ribosomes from the ORF1-deleted mutant, while it was restored by complementation of this strain.

Amino Acid Sequence↗

PubMed1995

Activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

Production of the antibiotic actinorhodin was activated in Streptomyces lividans under conditions in which it is not normally produced when transformed with an activator gene from S. lividans. The gene encodes a 86-nucleotide transcript, responsible for the actinorhodin production phenotype, which is homologous to the 132 nucleotide transcript from S. fradiae, thought to act as a putative antisense RNA.

Amino Acid Sequence↗

PubMed1994

Effect of glucose on agarase overproduction by Streptomyces.

The Streptomyces coelicolor dagA gene, coding for an extracellular agarase, has been propagated on a multicopy plasmid in S. coelicolor A3(2), the natural agarase producer strain and in S. lividans TK21, a closely related, nonproducer strain. The effect of the carbon source on the production of agarase by both strains, upon cultivation in liquid medium, revealed that the glucose repression affected the synthesis of agarase at the level of secretion, rather than at the level of transcription. In the presence of glucose, the pre-agarase was degraded intracellularly and the overall secretion of proteases decreased considerably in both strains, suggesting a negative regulatory role for glucose in the overall secretion in Streptomyces.

Blotting, Western↗

PubMed1994

Expression of an heterologous gene activating actinorhodin biosynthesis in Streptomyces lividans and Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin resulted in activation in the non-producer strain Streptomyces lividans, but not in the natural producer strain Streptomyces coelicolor, when transformed with an heterologous activator gene from Streptomyces fradiae. The gene encodes a 132 nucleotide-long transcript, responsible for the actinorhodin production phenotype, and thought to act as a putative antisense RNA, which has been detected in the transformed S. lividans cultures by reverse transcription followed by cyclic amplification.

Anthraquinones↗

PubMed1993

Heterologous recognition in vivo of promoter sequences from the Streptomyces coelicolor dagA gene.

The Streptomyces coelicolor dagA gene that codes for an extracellular agarase was cloned in the closely related bacterium S. lividans and transferred to the distantly related low G+C Gram-positive bacterium Bacillus subtilis and to the far more distantly related Gram-negative bacterium Escherichia coli. S1 nuclease mapping experiments identified a putative fifth promoter from which transcription of the dagA gene can take place, and accurately mapped the transcription termination site. The transcription terminator was specific for the Streptomyces strains and could terminate transcription initiated by promoters other than those of dagA. The agarase gene is efficiently transcribed in B. subtilis and E. coli, although pulse-chase experiments failed to detect the synthesis of agarase in these two bacteria.

Bacillus subtilis↗

PubMed1992

Heterologous activation of the actinorhodin biosynthetic pathway in Streptomyces lividans.

A DNA fragment of Streptomyces fradiae is able to activate the antibiotic actinorhodin biosynthetic pathway when cloned in Streptomyces lividans. The activator DNA region has been sequenced and its transcription initiation and termination sites accurately mapped in vivo. This DNA encodes a 132 nucleotides long transcript which is apparently responsible for the actinorhodin production phenotype, possibly acting as an antisense RNA. The sequence of the activator gene revealed no homology with any other known Streptomyces coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation.

Amino Acid Sequence↗

PubMed1992

Streptomyces lividans possesses a GroEL-like chaperonin.

Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin. This protein is specifically synthesized in bacterial cell cultures upon heat shock induction. It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E. coli. The S. lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E. coli and B. subtilis.

Bacterial Proteins↗

PubMed1991

Transcription of genes involved in the earliest steps of actinorhodin biosynthesis in Streptomyces coelicolor.

A 170bp long BamHI-Sau3A DNA fragment from the actIII-actI intergenic region of the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2) contains two promoters directing transcription in a divergent manner. One of them, the actIII promoter, is responsible for the transcription of the actIII gene and the other controls transcription of the adjacent actI region in the opposite direction. Weak activity of the actIII promoter can be detected in Streptomyces lividans and Bacillus subtilis in the absence but not in the presence of glucose. Neither promoter seems to function in Escherichia coli.

Anthraquinones↗

PubMed1989

Effects of internal deletions on the priming activity of the phage phi 29 terminal protein.

A series of internal deletions of gene 3, coding for the phage phi 29 DNA terminal protein, have been constructed and characterized. In addition, a substitution mutant in the sequence corresponding to amino acids (aa) 49-51 was obtained. The priming activity of the substitution mutant protein, in the formation of the protein p3-dAMP initiation complex, was drastically reduced suggesting that some of the aa present at position 49-51 are essential for p3 function. Deletions of 8 to 33 aa, from aa residue 48 towards the N terminus of the substitution mutant, further decreased the priming activity of the protein. The activity of deletion mutants lacking 15 or 21 aa from residue 57 towards the C terminus, and also containing a point mutation at position 56, was greatly reduced, and no activity was seen when 24 aa were lacking.

Amino Acid Sequence↗