José A. Gil

Porin expression in clinical isolates of Klebsiella pneumoniae.

Two porins, OmpK36 and OmpK35, have been described previously in Klebsiella pneumoniae, and they are homologous to the Escherichia coli porins OmpC and OmpF, respectively, at both the DNA and amino acid levels. Optimal resolution of the two K. pneumoniae porins by electrophoresis on polyacrylamide gels is not achieved using gel systems already described for E. coli and requires modifications of the bisacrylamide content of the resolving gels. Once resolved, identification of porins OmpK36 and OmpK35 cannot be based solely on their apparent molecular masses since in some strains the OmpK36 porin migrates faster than the OmpK35 porin, whilst in other strains OmpK35 is the faster-migrating porin. Expression of OmpK35 porin is increased in low-osmolarity medium and, combined with Western blot analysis, this allows for the identification of both porins. Application of this identification system showed that most isolates lacking expression of extended-spectrum beta-lactamases express the two porins, whereas most isolates producing these beta-lactamases express only porin OmpK36, and the OmpK35 porin is either very low or not expressed.

Characterization of an Endogenous Plasmid and Development of Cloning Vectors and a Transformation System in Brevibacterium lactofermentum

A cryptic plasmid, pBL1 of 4.3 kb, has been found in lysine-producing Brevibacterium lactofermentum strains BL0, BL70, BL74 and BL77. pBL1 had single restriction sites for BalI, BclI, HaeII, HindIII and HpaI. It had four sites for AvaI, seven for HaeIII, eight for MboI and a very large number for AluI, but no sites were found for PstI, EcoRI or BamHI. The estimated copy number was 30. Three different pBL1-pBR322 hybrids named pUL1, pUL10 and pUL20 were constructed. Transposon Tn5 was inserted by transposition into either the pBR322 or the pBL1 components of plasmid pUL1, pUL10 and pUL20. A shuttle vector able to replicate in Escherichia coli, Streptomyces lividans and B. lactofermentum was constructed by cloning pBL1 into the plasmid pIJ860, a bifunctional E. coli-S. lividans vector carrying the tsr, bla and kan genes. A polyethylene glycol-assisted transformation system for B. lactofermentum protoplasts was developed. Transformation frequencies of 102 transformants (μg DNA)−1 were obtained. The kan resistance gene from Tn5 was expressed very efficiently in B. lactofermentum (up to 200 μg ml−1). A smaller plasmid, pUL62, was constructed in which the tsr (thiostrepton resistance) gene of pUL61 was deleted.

Analysis of Genes Involved in Arsenic Resistance in Corynebacterium glutamicum ATCC 13032

ABSTRACT Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1′) located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1′, arsR2, arsB2, and arsC2 being inducible by arsenite.

Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped Actinomycete Corynebacterium glutamicum

The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA(Cg)) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA(Cg)-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA(Cg) localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

Assemblies of DivIVA Mark Sites for Hyphal Branching and Can Establish New Zones of Cell Wall Growth in Streptomyces coelicolor

Time-lapse imaging of Streptomyces hyphae revealed foci of the essential protein DivIVA at sites where lateral branches will emerge. Overexpression experiments showed that DivIVA foci can trigger establishment of new zones of cell wall assembly, suggesting a key role of DivIVA in directing peptidoglycan synthesis and cell shape in Streptomyces.

Arsenate reductase, mycothiol, and mycoredoxin concert thiol/disulfide exchange.

We identified the first enzymes that use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defense against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unraveled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin, a redox enzyme for which the function was never shown before, reduces the thiol-arseno bond and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ∼5 m-1 s-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in actinobacteria.

The candicidin gene cluster from Streptomyces griseus IMRU 3570

A 205 kb DNA region from Streptomyces griseus IMRU 3570, including the candicidin biosynthetic gene cluster, was cloned and partially sequenced. Analysis of the sequenced DNA led to identification of genes encoding part of a modular polyketide synthase (PKS), genes for thioesterase, macrolactone ring modification, mycosamine biosynthesis and attachment to the macrolide ring, candicidin export and regulatory proteins. It represents the first extensive genetic characterization of an aromatic polyene macrolide antibiotic biosynthetic gene cluster. Of particular interest is the presence of the CanP1 loading domain (the first described as responsible for the activation of an aromatic starter unit) and the polypeptide CanP3 (carrying modules for the formation of five out of seven conjugated double bonds). Disruption of the pabAB gene that encodes the starter unit of candicidin abolished its production [which was restored when exogenous p-aminobenzoic acid (PABA) was supplied to the culture] and resulted in an enhanced production of another antifungal compound that is barely detected in the wild-type.

Sporulation of Several Species of Streptomyces in Submerged Cultures after Nutritional Downshift

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.

Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.