Branko Jovcic

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.

Pseudomonas sp. strain ATCC19151 is a natural isolate from sewage with the ability to degrade detergents. Genes encoding potential choline sulfatase (betC), substrate-binding ABC transporter protein (betD), sulfate transporter (betE), and divergent putative transcriptional regulator (betR) were cloned and characterized from strain ATCC19151. In silico analysis revealed that (1) the BetC protein belongs to alkPPc superfamily and shares CXPXR sequence with the cysteine sulfatases of group I, (2) BetR belongs to the LysR family of transcriptional regulators, (3) BetD is part of the PBPb superfamily of periplasmic and membrane-associated proteins, and (4) BetE is a permease and contains STAS domain. Insertional mutagenesis and genetic complementation show that betC gene encodes a functional choline sulfatase. Analysis of the betC (P betC ) and betR (P betR ) promoters revealed that they are inducible. BetR activates betC and betR transcription in the presence of choline sulfate, whilst in the absence of choline sulfate, BetR represses its own transcription. It was further established that BetR directly binds to betC–betR intergenic region in vitro, with higher affinity in the presence of choline sulfate as cofactor. Transcription of betC and betR was not induced in the presence of high concentration of NaCl.

Ten lactobacilli and one leuconostoc showing auto-aggregation ability were isolated from artisanal cheeses. Furthermore, non-aggregation strains were isolated from the same cheese sample, if existed. The analysis of factor(s) possibly involved in auto-aggregation was performed. The pretreatment of cells with proteinase K resulted in the disappearance of auto-aggregation ability. Moreover, cells also lost aggregation ability after three-times, successive washing in distilled water. Testing the ability of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 and its aggregation-deficient derivative BGSJ2-81 to co-aggregate with Listeria innocua ATCC33090, Escherichia coli ATCC25922 or Salmonella typhimurium TR251 showed that strain BGSJ2-8 co-aggregated with these strains, but derivative BGSJ2-81 was not. However, the treatment of L. paracasei subsp. paracasei BGSJ2-8 with proteinase K prior to co-aggregation tests resulted in losing co-aggregation ability. Surface properties of selected strains were analyzed by MATS (microbial adhesion to solvents) method. It was noticed that the strains with auto-aggregation ability were highly hydrophobic in comparison with aggregation-deficient ones. Comparative analyses of the surface features of strain L. paracasei subsp. paracasei BGSJ2-8 and its derivative BGSJ2-81 revealed notable difference.

Aims:  The presented study was aimed to reveal transcriptional regulation of genes involved in SDS degradation (sdsA and sdsB) in Pseudomonas sp. ATCC19151. In addition, the ability of Pseudomonas sp. ATCC19151 to degrade anionic surfactants present in commercial detergent and septic tank drain was analysed.Methods and Results:  Strain ATCC19151, at 30°C, degrades all SDS present in the liquid medium (up to 4% w/v of SDS) within 48 h. ATCC19151 grows in the presence up to 15% (v/v) ‘Fairy’ commercial detergent and mineralizes 35% of present anionic surfactants. Analysis of the sdsA (PsdsA) and divergent sdsB (PsdsB) gene promoter activities revealed that SdsB acts as a positive regulator of sdsA and sdsB transcription. PsdsA and PsdsB activities rose significantly in the presence of the SDS, indicating inducibility of sdsA and sdsB transcription. DNA-binding assay indicated that SdsB directly regulates the transcription of sdsA and sdsB genes. Strain ATCC19151 grew in a sterile septic tank drain and on commercial detergent as sole source of carbon.Conclusions:  SdsA enables Pseudomonas sp. ATCC19151 to utilize SDS as a sole carbon source. SdsB is positive transcriptional regulator of sdsA and sdsB genes.Significance and Impact of the Study:  Ability of ATCC19151 to degrade anionic surfactants makes Pseudomonas sp. ATCC19151 a good candidate for bioremediation.

The σS subunit of RNA polymerase is a central regulator which governs the expression of a host of stationary phase-induced and osmotically regulated genes in Gram-negative bacteria. The Pseudomonas putida rpoS gene is transcribed as a monocistronic rpoS mRNA with a 368 nucleotide-long 5′ untranslated region (5′ UTR). In this study, we investigate the posttranscriptional control of RpoS synthesis using rpoS-lacZ transcriptional and translational fusions consisting of the native promoter and deletions of 5′ UTR or insertion into UTR. The differing activity of constructed translational fusions strongly indicated that the 5′ UTR is involved in the translational regulation of RpoS expression in the stationary phase. The results obtained herein demonstrated that the structure of UTR performs an important function in the translational regulation of the rpoS gene.