Co-utilization of saccharides in mixtures: Moving toward a new understanding of carbon metabolism in Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used in food production, notably in yogurt fermentation. It evolved under highly specific ecological conditions, resulting in its ability to efficiently metabolize lactose, the main saccharide in milk. However, when used in sweetened dairy products or plant-based products, S. thermophilus may encounter other saccharides (i.e. alone or in mixtures). To date, the bacterium's metabolic capacities in such contexts have been poorly characterized. Here, we explored saccharide utilization by 39 S. thermophilus strains. Using in silico analysis, we discovered that the identity and structure of saccharide utilization genes are conserved across strains, and we identified six saccharides that might be metabolized. Although underlying genetic variability was low, strains nonetheless displayed differences in growth when supplied with different saccharides: lactose, sucrose, fructose, and glucose. Interestingly, we found that strains preferentially used lactose and sucrose in tandem when given saccharide mixtures. Furthermore, we uncovered some main potential drivers of saccharide metabolism in S. thermophilus. Notably, the sucrose transporter ScrA is also responsible for importing glucose. Overall, this research has yielded useful findings that can help the development of new fermented foods, including plant-based products, in which sucrose may serve as a major carbon source.

Physiological study of Lactobacillus delbrueckii subsp. bulgaricus strains in a novel chemically defined medium.

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

Random and directed mutagenesis to elucidate the functional importance of helix II and F-989 in the C-terminal secretion signal of Escherichia coli hemolysin.

The HlyA secretion signal sequence of approximately 46 residues is predicted to contain helix I and an amphipathic helix II separated by a short loop including the conserved Phe residue, F-989. All nine substitutions of Phe-989 drastically reduce secretion of HlyA. Directed mutagenesis identified a functional hot spot, EISK, in helix II. However, genetic analysis did not provide strong support for a functional helix II; rather, the results emphasized that individual residues, for example, E-978 and F-989, are essential irrespective of a specific secondary structure.

Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway.

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.

Protein secretion pathway in Escherichia coli.

The export of proteins to the Escherichia coli periplasm is a well established system for heterologous protein production. With a better understanding of the protein export (SecA, Y-dependent) process and a greater awareness of the conditions necessary for correct folding of proteins in the periplasm, serious efforts are now being made to manipulate this system to achieve substantial increases in the yield of authentically folded proteins. Further advances in the development of methods for the recovery of recombinant proteins from the culture medium have made the use of fusion proteins secreted by the protein A or haemolysin pathways a more attractive option. Recent studies of the haemolysin system indicate its ability to secrete a wide range of polypeptides, including normally cytoplasmic proteins. As their features and potential applications become much clearer, a rapidly expanding number of protein-secretion mechanisms in Gram-negative bacteria are becoming available for heterologous protein expression. Most, if not all, of these systems can be successfully transplanted into E. coli, providing a wider choice of systems for the future.

Evidence that residues -15 to -46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator.

We previously identified three well-dispersed mutations, E978-K, F989-L and D1009-R within the haemolysin A signal region, located at positions -46, -35 and -15, with respect to the C-terminus, respectively. Each mutation reduces the efficiency of secretion two- to threefold leaving 30-45% of the wild-type activity. We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations. The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild-type signal. To test this further, we developed a secretion competition assay in order to classify signal mutations. We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild-type HlyA co-expressed in the same cell. The properties of the fusion indicate that it blocks the translocator. The three mutations singly and in combinations were recombined in vitro into the 3'-end of the hybrid gene. In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HlyA. All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium. This would indicate that residues involved in recognition (or steps leading to recognition) extend from at least -15 to -46 with respect to the HlyA C-terminus.