The gene cluster of aureocyclicin 4185: the first cyclic bacteriocin of Staphylococcus aureus.

Staphylococcus aureus 4185 was previously shown to produce at least two bacteriocins. One of them is encoded by pRJ101. To detect the bacteriocin-encoding gene cluster, an ~9160 kb region of pRJ101 was sequenced. In silico analyses identified 10 genes (aclX, aclB, aclI, aclT, aclC, aclD, aclA, aclF, aclG and aclH) that might be involved in the production of a novel cyclic bacteriocin named aureocyclicin 4185. The organization of these genes was quite similar to that of the gene cluster responsible for carnocyclin A production and immunity. Four putative proteins encoded by these genes (AclT, AclC, AclD and AclA) also exhibited similarity to proteins encoded by cyclic bacteriocin gene clusters. Mutants derived from insertion of Tn917-lac into aclC, aclF, aclH and aclX were affected in bacteriocin production and growth. AclX is a 205 aa putative protein not encoded by the gene clusters of other cyclic bacteriocins. AclX exhibits 50 % similarity to a permease and has five putative membrane-spanning domains. Transcription analyses suggested that aclX is part of the aureocyclicin 4185 gene cluster, encoding a protein required for bacteriocin production. The aclA gene is the structural gene of aureocyclicin 4185, which shows 65 % similarity to garvicin ML. AclA is proposed to be cleaved off, generating a mature peptide with a predicted Mr of 5607 Da (60 aa). By homology modelling, AclA presents four α-helices, like carnocyclin A. AclA could not be found at detectable levels in the culture supernatant of a strain carrying only pRJ101. To our knowledge, this is the first report of a cyclic bacteriocin gene cluster in the genus Staphylococcus.

Genes involved in immunity to and secretion of aureocin A53, an atypical class II bacteriocin produced by Staphylococcus aureus A53.

Aureocin A53 is an antimicrobial peptide produced by Staphylococcus aureus A53. The genetic determinants involved in aureocin A53 production and immunity to its action are organized in at least four transcriptional units encoded by the 10.4-kb plasmid pRJ9. One transcriptional unit carries only the bacteriocin structural gene, aucA. No immunity gene is found downstream of aucA, as part of the same transcriptional unit. Further downstream of aucA is found an operon which contains the three genes aucEFG, whose products seem to associate to form a dedicated ABC transporter. When aucEFG were expressed in RN4220, an aureocin A53-sensitive S. aureus strain, this strain became partially resistant to the bacteriocin. A gene disruption mutant in aucE was defective in aureocin A53 externalization and more sensitive to aureocin A53 than the wild-type strain, showing that aucEFG are involved in immunity to aureocin A53 by active extrusion of the bacteriocin. Full resistance to aureocin A53 was exhibited by transformants carrying, besides aucEFG, the operon formed by two genes, aucIB and aucIA, located between aucA and aucEFG and carried in the opposite strand. AucIA and AucIB share similarities with hypothetical proteins not found in the gene clusters of other bacteriocins. A gene disruption mutant in orf8, located upstream of aucA and whose product exhibits about 50% similarity to a number of hypothetical membrane proteins found in many Gram-positive bacteria, was strongly affected in aureocin A53 externalization but resistant to aureocin A53, suggesting that Orf8 is also involved in aureocin A53 secretion.

Hyicin 3682, a bioactive peptide produced by Staphylococcus hyicus 3682 with potential applications for food preservation.

Bacteriocins are peptides produced by bacteria and having inhibitory activity against other bacteria. Many of these substances may be useful as antibacterial agents for practical applications. In this study, 21 Staphylococcus spp. isolated from pigs, dogs and bovine milk in different states of Brazil were investigated for staphylococcin production. Hyicin 3682, a bacteriocin produced by one such strain, inhibited almost all strains tested, including Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. PCR experiments showed that hyicin 3682 is lantibiotic-related, but not identical, to both epidermin and Bsa. The maximum production of hyicin 3682 (6,400 AU/ml) was observed after 24 h of growth in BHI medium at 37 °C. Hyicin 3682 proved to be a cationic, small antimicrobial peptide with a molecular mass of 2,139 Da. It exhibited resistance to low pH and to heating at 65 °C, and partial sensitivity to proteolytic enzymes. Taken together, these results suggest that hyicin 3682, the first bacteriocin characterized in Staphylococcus hyicus, has potential biotechnological applications as a food preservative. Moreover, hyicin 3682 was able to inhibit its producer strain, suggesting that an effective immune system for specific protection against hyicin 3682 is not found in its producer strain, a characteristic not described thus far for other staphylococcins.

Revealing the latent mobilization capability of the staphylococcal bacteriocinogenic plasmid pRJ9.

Plasmid pRJ9 is a non-self-mobilizable bacteriocinogenic plasmid from Staphylococcus aureus. Despite this feature, DNA sequencing and RT-PCR experiments showed that it presents a Mob region with three genes (mobCAB), transcribed as an operon. In silico analysis of the Mob proteins encoded by pRJ9 showed that they present all the conserved functional features reported until present as being essential for plasmid mobilization. Moreover, they showed a high identity to Mob proteins encoded by mobilizable plasmids from Staphylococcus spp., especially to those encoded by plasmid pRJ6, which presents four mob genes (mobCDAB). A putative oriT region was also found upstream of the pRJ9 mob operon. pRJ9 could only be successfully mobilized by pGO1 when pRJ6 was present in the same strain. Further experiments showed that the pRJ9 oriT can be recognized by the pRJ6 Mob proteins, confirming its functionality. As pRJ9 does not possess a mobD gene while pRJ6 does, the absence of this gene was believed to be responsible for its lack of mobilization. However, conjugation experiments with a donor strain carrying also mobD cloned into an S. aureus vector showed that pRJ9 does not become mobilized even in the presence of the protein MobD encoded by pRJ6. Therefore, the reasons for pRJ9 failure to be mobilized are presently unknown.

Nukacin 3299, a lantibiotic produced by Staphylococcus simulans 3299 identical to nukacin ISK-1.

Nukacin 3299 (formerly designated simulancin 3299), produced by a Staphylococcus simulans strain involved in bovine mastitis in Brazil, is the first peptide bacteriocin described in this staphylococcal species. With the intent to elucidate some aspects of its biology, nukacin 3299 was purified and characterized. The mass of the purified bacteriocin was shown to be 2957.3 Da, and the peptide N-terminal amino acids (KKKSGVI) were identified by Edman degradation. The nukacin 3299 structural gene, nukA, was detected by PCR and DNA sequencing, showing that this bacteriocin is identical to nukacin ISK-1, a 27-amino acid type-A (II) lantibiotic produced by Staphylococcus warneri ISK-1, isolated from a "nukadoko", in Japan. The genes involved in nukacin 3299 biosynthesis are located on plasmid pRJ97 (>27 kb). They have an organization similar to that of the nukacin ISK-1 gene cluster, excepted for the presence of an IS257/431 element (791 bp) present between the orf1 and nukA genes of the nukacin 3299 gene cluster. The presence of this insertion sequence is expected to affect the expression of orf1, whose function is presently unknown. Nukacin 3299 proved to be sensitive to proteolytic enzymes and relatively stable at different temperatures and between pH 3.0-9.0. Nukacin 3299 exhibited activity towards staphylococcal strains involved in bovine mastitis, showing a potential application on mastitis control, a disease with great economic impact.

Aureocins 4185, bacteriocins produced by Staphylococcus aureus 4185: potential application in food preservation.

In the present study, the bacteriocins produced by Staphylococcus aureus 4185, a strain isolated from bovine mastitis, were purified and partially characterized. After purification by ammonium sulfate precipitation, cation-exchange chromatography, and five runs of high-performance liquid chromatography (HPLC), antimicrobial activity was recovered with 40% and 80% isopropanol, suggesting that more than one antimicrobial peptide, named aureocins 4185, is produced by S. aureus 4185. Mass spectrometry analyses revealed three peptides eluted with 40% isopropanol: peptide A (2,305.3 +/-1.5 Da), peptide B (2,327.3 +/-1.5 Da), and peptide C (3,005.5 +/-1.5 Da), and two peptides eluted with 80% isopropanol: peptide D (6,413.5 +/-1.5 Da) and peptide E (12,834.5 +/-1.5 Da). Although five peptides have been detected, only four small peptide sequences were obtained by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)/TOF mass spectrometry analyses: SLLEQFTGK (eluted with 40% isopropanol), ALLYDER, NNTSHNLPLGWFNVK, and NNLAQGTFNATK (eluted with 80% isopropanol). The sequences SLLEQFTGK and ALLYDER revealed identity with hypothetical peptides with unknown function. The sequences NNTSHNLPLGWFNVK and NNLAQGTFNATK showed similarity to a segment of a precursor of staphylococcal autolysins. The antimicrobial activity detected in the supernatant of strain 4185 proved to be resistant to heat treatment at 65°C; however, treatment at 80°C abolished completely its antimicrobial properties. The concentrated supernatant containing aureocins 4185 exhibited a strong bacteriolytic activity toward Micrococcus luteus ATCC 4698. Additionally, aureocins 4185 exhibited antagonistic activity against important foodborne pathogens, including Listeria monocytogenes, thus showing a potential application in food preservation.

Bacteriocin production by Staphylococcus aureus involved in bovine mastitis in Brazil.

In the present study, 257 Staphylococcus spp. strains were isolated from bovine mastitis cases in 56 different Brazilian dairy herds located in the southeast region of the country and tested for antimicrobial substance (AMS) production. Forty-six strains (17.9%) exhibited AMS production and their identification as Staphylococcus aureus was based on the presence of Gram-positive cocci and on positive results in tests for the ability to coagulate rabbit plasma, to ferment mannitol, and to produce acetoin. The AMS were characterized as bacteriocins (Bac) by their sensitivity to proteolytic enzymes. The Bac(+) strains were tested for resistance to 14 antimicrobial agents showing different profiles. Eighteen strains (39.0%) expressed a multiple antibiotic resistance phenotype. Forty-five strains exhibited at least one plasmid DNA. Cross-immunity analysis against strain S. aureus A70, which produces aureocin A70, amplification of the aurABCD operon (which encodes aureocin A70) or detection of this same operon by DNA/DNA hybridization revealed that 34 strains produce bacteriocins either identical or similar to aureocin A70. The remaining 12 Bac(+) strains produce antimicrobial peptides that seem to be distinct from the best characterized staphylococcal bacteriocins described thus far. The bacteriocin produced by strain 4185 may possess potential practical applications, since it was able to inhibit important pathogens such as Bacillus cereus, Listeria monocytogenes, and Staphylococcus spp. isolated from nosocomial infections.

The gene bap, involved in biofilm production, is present in Staphylococcus spp. strains from nosocomial infections.

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii.

Mobilization functions of the bacteriocinogenic plasmid pRJ6 of Staphylococcus aureus.

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT (pRJ6) was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT (pRJ6), is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT (pRJ6) of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra(pC223) site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.

Genomic fingerprinting of bacteriocin-producer strains of Staphylococcus aureus.

Among 363 strains of Staphylococcus aureus, 21 were shown to produce bacteriocins (Bac), antimicrobial peptides with potential biotechnological applications. This collection includes strains which are either isolated from food, patients and healthy cattle, or are involved in subclinical bovine mastitis. From these 21 strains, 17 were shown to carry closely-related 8.0-kb Bac plasmids encoding bacteriocins either identical to or similar to aureocin A70, a bacteriocin able to inhibit strains of Listeria monocytogenes, a food-borne pathogen. Such findings prompted us to investigate the genetic relationships among these Bac+ strains. To obtain more discriminatory results, a combined analysis of AP-PCR, rep-PCR, and a modified PCR technique that we designated SD-PCR was employed. The 17 Bac+ strains harboring 8.0-kb Bac plasmids exhibited seven fingerprint patterns. One such genotype was composed of 8 out of the 11 strains associated with bovine mastitis, which suggests the prevalence of a clone of Bac+ strains involved in this animal infection carrying 8.0-kb Bac plasmids. Our data support the assumption that Bac+ strains of S. aureus carrying genetically related 8.0-kb Bac plasmids do not belong to a single clone. It seems, therefore, that 8.0-kb Bac plasmids have spread horizontally among different S. aureus strains. There also seems to be genetic diversity among the remaining Bac+ strains analyzed.