Piersandro Cocconcelli

High frequency of conjugation in Lactobacillus mediated by an aggregation-promoting factor

Summary: Lactobacillus plantarum strain 4B2, which exhibits a strong autoaggregating phenotype, receives the broad-host-range plasmid pAMβ1 with conjugation efficiencies as high as 10-2 transconjugants per donor using solid matings; broth matings also occur, but at low transfer frequencies. Filter-sterilized spent supernatant of this strain contains a 32 kDa protein that promotes aggregation, and consequently a high frequency of conjugation, in lactic acid bacteria containing α-1,2-glucose-substituted lipoteichoic or teichoic acids. It appears, therefore, that the substituted lipoteichoic or teichoic acids act as receptors for the aggregation-promoting protein.

Contribution of enterococci to the spread of antibiotic resistance in the production chain of swine meat commodities.

Thirty-six samples, including fecal specimens, dry feedstuffs, raw and processed pork meat products, and dry fermented sausages, were collected from two production chains of swine meat commodities and analyzed for the presence of 11 antibiotic resistance (AR) genes. Specific PCR assays carried out on DNA extracted directly from the samples revealed a high incidence of the genes tet(K) (80.5%), ermB (66.7%), and tet(M) (66.7%). Feces and feedstuffs gave the largest number of positive amplifications. To elucidate the contribution of enterococci to the occurrence and spread of AR, 146 resistant enterococci were isolated, and their identity, genetic fingerprints, and AR gene profiles were determined by means of molecular techniques. Enterococcus faecalis and Enterococcus faecium were the predominant isolated species (43.8 and 38.4%, respectively); Other Enterococcus species identified were E. durans (8.9%), E. hirae (2.7%), E. gallinarum (2.1%), E. mundtii (2.1%), and E. casseliflavus (2.1%). A number of isolates displayed a complex AR gene profile comprising up to four different resistance determinants. The genes tet(M) and ermB were highly diffused, being present in 86.9 and 84.9%, respectively, of the isolates. The application of amplified fragment length polymorphism fingerprinting was particularly valuable to monitor the resistant enterococcal isolates along the production chain and to individuate steps in which contamination might occur. In fact, isolates of E. faecalis and E. faecium showing the same amplified fragment length polymorphism profile and AR gene pattern were detected in samples taken at different steps of the food chain suggesting three cases of bacterial clonal spread.

Single-stranded DNA plasmid, vector construction and cloning of Bacillus stearothermophilus α-amilase in Lactobacillus

Vector plasmids were constructed by ligating chloramphenicol and erythromycin resistance genes to TaqI-digested DNA of a cryptic plasmid from Lactobacillus plantarum. The minimal region of Lactobacillus plasmid DNA that was required for DNA replication was defined and a single-stranded DNA intermediate replication system was observed. Homologies with other origins of replication of plasmids from Gram-positive bacteria, replicating via rolling circle mechanism, were found. It was shown that the constructed vectors, named pPSC20 and pPSC22, were transformable into L. plantarum, Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus helveticus, Lactococcus lactis subsp. lactis, Bacillus subtilis, and Escherichia coli. Using plasmid pPSC22, the alpha-amylase gene of Bacillus stearothermophilus was cloned and expressed in several Lactobacillus species.

Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22

The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.

Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS

ABSTRACT In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment. IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Purification of Lactobacillus secreted proteins

We describe a rapid and reliable one-step method for purification of Lactobacillus secreted proteins. With electroendosmotic preparative electrophoresis and a modified synthetic medium the L. plantarum aggregation promoting factor, a 32 kDa secreted protein, and the thermostable α-amylase of Bacillus stearothermophilus cloned in L. reuteri were purified. Although the growth rate was reduced, the production of secreted proteins was not affected.

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg2+-ATP as a function of K+ concentration (2–200 mM). Mg2+-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K+, while it amounted to 6 nmol ATP hydrolyzed min–1 (mg membrane protein)–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K+-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (Km = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-32P]ATP in the presence of K+ and La3+ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K+ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation.