Biomedical subjects

Saulius Kulakauskas

Publications and source records attributed to Saulius Kulakauskas.

8 recordsLinked to original sources

PubMed2025

Lactococcal mobile genetic elements harbour a diverse phage defensome rich in restriction-modification systems.

The genomes of 43 distinct lactococcal strains were reconstructed by a combination of long- and short-read sequencing, resolving the plasmid complement and methylome of these strains. The genomes comprised 43 chromosomes of approximately 2.5 Mb each and 269 plasmids ranging from 2 to 211 kb (at an average occurrence of 6 per strain). A total of 953 antiphage genes representing 538 phage defence systems were identified in the 43 strains and were catalogued and cross-correlated with co-occurrent mobile elements, which indicated that almost 60% of these systems are predicted to be mobile. Detailed analysis established that restriction-modification (R-M) systems form a significant portion of this mobile phage defensome. As such, all detected Type I, II, and III-associated methylated motifs (46 of which were unique to this study) were matched to their corresponding methylating enzymes by homology detection or molecular cloning. The cumulative antiphage activity of selected systems and the ability of truncated R-M genes to contribute to methylation were demonstrated. This study reveals, for the first time, the dairy lactococcal plasmidome to be a rich reservoir of orphan HsdS-encoding genes, in a comprehensive survey of (mobile) phage defence systems in lactic acid bacteria.

Bacteriophages↗

PubMed2006

Identification of an essential gene responsible for D-Asp incorporation in the Lactococcus lactis peptidoglycan crossbridge.

Bacteria such as Lactococcus lactis have D-aspartate (D-Asp) or its amidated derivative D-asparagine (D-Asn), in their peptidoglycan (PG) interpeptide crossbridge. We performed a subtractive genome analysis to identify L. lactis gene yxbA, orthologues of which being present only in bacteria containing D-amino acids in their PG crossbridge, but absent from those that instead insert L-amino acids or glycine. Inactivation of yxbA required a complementing Streptococcus pneumoniae murMN genes, which express enzymes that incorporate L-Ser-L-Ala or L-Ala-L-Ala in the PG crossbridge. Our results show that (i) yxbA encodes D-Asp ligase responsible for incorporation of D-Asp in the PG crossbridge, and we therefore renamed it as aslA, (ii) it is an essential gene, which makes its product a potential target for specific antimicrobials, (iii) the absence of D-Asp may be complemented by L-Ser-L-Ala or L-Ala-L-Ala in the L. lactis PG, indicating that the PG synthesis machinery is not selective for the side-chain residues, and (iv) lactococcal strains having L-amino acids in their PG crossbridge display defects in cell wall integrity, but are able to efficiently anchor cell wall proteins, indicating relative flexibility of lactococcal transpeptidation reactions with respect to changes in PG sidechain composition.

Aspartic Acid↗

PubMed2006

Peptidoglycan structure analysis of Lactococcus lactis reveals the presence of an L,D-carboxypeptidase involved in peptidoglycan maturation.

Detailed structural analysis of Lactococcus lactis peptidoglycan was achieved by identification of its constituent muropeptides separated by reverse phase high-performance liquid chromatography. Modification of the classical elution buffer allowed direct and sensitive analysis of the purified muropeptides by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The structures of 45 muropeptides were assigned for L. lactis strain MG1363. Analysis of the muropeptide composition of an MG1363 dacB mutant showed that the dacB-encoded protein has l,d-carboxypeptidase activity and is involved in peptidoglycan maturation.

Base Sequence↗

PubMed2005

Immobilization-based isolation of capsule-negative mutants of Streptococcus pneumoniae.

The capsular polysaccharide (CPS) is the most important identified virulence factor of Streptococcus pneumoniae, a human pathogen of the upper respiratory tract. One limitation in studies of S. pneumoniae surface virulence factors is the lack of a reliable procedure for isolation of capsule-negative mutants of clinical strains. This paper presents an approach, based on the immobilization of pneumococci in semi-liquid (0.04 % agar) medium, to easily distinguish and select for non-capsulated mutants. A clinical S. pneumoniae type 37 strain was used as a model to show that CPS production results in bacterial immobilization in semi-liquid agar medium and restricts cell sedimentation. Descendants of CPS(-) mutants sedimented faster under these conditions and therefore could be separated from immobilized parental cells. The CPS(-) phenotype of the obtained mutants was confirmed by both immunoagglutination and immunostaining experiments using specific type 37 capsular antibodies. Complementation of immobilization with the cloned tts gene, encoding type 37 CPS synthase, confirmed that faster sedimentation of mutants was specifically due to loss of the capsule. DNA sequence determination of three independent mutants revealed a point mutation, a 46 nt deletion and a heptanucleotide duplication in the tts gene. Immobilization of strains producing other CPSs (type 2, 3 and 6) also resulted in the appearance of CPS(-) mutants, thus showing that immobilization-based isolation is not restricted to type 37 pneumococci. Bacterial growth in semi-liquid medium proved to be a useful model system to identify the genetic consequences of immobilization. The results indicate that immobilization due to CPS may impose selective pressure against capsule production and thus contribute to capsule plasticity.

Agglutination Tests↗

PubMed2004

Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.

Bacterial Proteins↗

PubMed2002

Positive role of peptidoglycan breaks in lactococcal biofilm formation.

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Gram-positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild-type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain-forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm-forming capacity. Direct selection for strains with increased biofilm-forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.

Bacterial Adhesion↗

PubMed2002

N4,5-dimethylcytosine, a novel hypermodified base in DNA.

Successive enzymatic modification of DNA by two methyltransferases that recognize identical or overlapping sequences and normally target either 5- or N4-position of the same cytosine residue, can lead to the formation of a doubly methylated base--N4,5-dimethylcytosine. Implications of such enzymatic "permethylation" of cytosine on the structure and interactions of DNA are examined.

Cytosine↗

PubMed2002

Respiration capacity and consequences in Lactococcus lactis.

We recently reported that the well-studied fermenting bacterium Lactococcus lactis could grow via a respirative metabolism in the presence of oxygen when a heme source is present. Respiration induces profound changes in L. lactis metabolism, and improvement of oxygen tolerance and long-term survival. Compared to usual fermentation conditions, biomass is approximately doubled by the end of growth, acid production is reduced, and large amounts of normally minor end products accumulate. Lactococci grown via respiration survive markedly better after long-term storage than fermenting cells. We suggest that growth and survival of lactococci are optimal under respiration-permissive conditions, and not under fermentation conditions as previously supposed. Our results reveal the uniqueness of the L. lactis respiration model. The well-studied 'aerobic' bacteria express multiple terminal cytochrome oxidases, which assure respiration all throughout growth; they also synthesize their own heme. In contrast, the L. lactis cydAB genes encode a single cytochrome oxidase (bd), and heme must be provided. Furthermore, cydAB genes mediate respiration only late in growth. Thus, lactococci exit the lag phase via fermentation even if heme is present, and start respiration in late exponential phase. Our results suggest that the spectacularly improved survival is in part due to reduced intracellular oxidation during respiration. We predict that lactococcal relatives like the Enterococci, and some Lactobacilli, which have reported respiration potential, will display improved survival under respiration-permissive conditions.

Electron Transport Complex IV↗