S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence

Vidgrén, Gabriele; Palva, Ilkka; Pakkanen, Raimo; Lounatmaa, Kari; Palva, Airi

doi:10.1128/jb.174.22.7419-7427.1992

Cited by 129 publications

(144 citation statements)

References 37 publications

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“…In keeping with other S-layer subunit proteins (18,29,43,45), the vapA transcript expressed from the P1 promoter was found to be monocistronic. Indeed, only the Bacillus S-layers have been shown to be polycistronic (1).…”

Section: Discussionsupporting

confidence: 52%

“…Several other S-layer systems have multiple promoters; in these cases, however, primer extension experiments have shown that these promoters are active during growth in vitro. For example, three transcriptional start sites have been shown in front of the structural genes for the S-layers of Bacillus brevis 47, the P2 and P3 promoters being most active (1), while the Lactobacillus brevis S-layer protein gene possesses two promoters which appear to be equally active during exponential growth phase (45).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, the presence of a long mRNA leader appears to be a characteristic of certain S-layer subunit protein mRNAs. For example, the constitutive P2 promoter of the B. brevis 47 cwp operon provides for a 247-bp transcriptional leader, and 111-to 130-bp transcriptional leaders have been reported for other S-layer genes (16,45). Long mRNA leaders are also seen in attenuated operons such as the amino acid biosynthetic operons (29).…”

Section: Discussionmentioning

confidence: 99%

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Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA

et al. 1993

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The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer. Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences. Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A. salmonicida. Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A. salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures. Northern analysis of cells grown at 15°C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25). At 15°C, the half-life of the vapA mRNA was 22 min, while at 20°C, the half-life was significantly shorter, 11 min. The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin. Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA. vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein.

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Section: Discussionsupporting

confidence: 52%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA

et al. 1993

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“…The secretion probe vector is based on the signal peptide sequence of the L. brevis surface layer protein SlpA (43) and the nuc gene for Staphylococcus aureus nuclease (37), devoid of its export signal (⌬ SP nuc). The SP slpA signal sequence (with PstI and AatII restriction sites added at the 5Ј and 3Ј ends) was amplified using the primers shown in Table 2, using chromosomal DNA of L. brevis as template.…”

Section: Methodsmentioning

confidence: 99%

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Gaeng

Scherer

Neve³

et al. 2000

Appl Environ Microbiol

132

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Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SP slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SP slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3 -end coding sequence of SP slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511⌬(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SP SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511⌬C into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

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“…To determine the correlation between the level of expression and codon usage preference in lactobacilli, we have compared the cumulative codon usage values of NNC/U-type and NNG/A-type two-codon sets ofLactobacillus genes known to be efficiently expressed [e.g. S-layer proteins constitute 5-10% of total proteins (27,28), L-LDH represents >2% of soluble protein in lactobacilli (29)] with those of genes known or presumed to be inefficiently expressed (e.g. repA gene of plasmid p353-2, and xylB and xylR genes of L.pentosus (21,30), and the regulatory gene lacT from L. casei (31).…”

Section: Codon Usage In Lactobacillusmentioning

confidence: 99%

Divergence in codon usage ofLactobacillus species

Pouwels¹,

Leunissen

1994

Nucl Acids Res

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We have analyzed codon usage patterns of 70 sequenced genes from different Lactobacillus species. Codon usage in lactobacilli is highly biased. Both inter-species and intra-species heterogeneity of codon usage bias was observed. Codon usage in L.acidophilus is similar to that in L.helveticus, but dissimilar to that in L.bulgaricus, L.casei, L.pentosus and L.plantarum. Codon usage in the latter three organisms is not significantly different, but is different from that in L.bulgaricus. Inter-species differences in codon usage can, at least in part, be explained by differences in mutational drift. L.bulgaricus shows GC drift, whereas all other species show AT drift. L.acidophilus and L.helveticus rarely use NNG in family-box (a set of synonymous) codons, in contrast to all other species. This result may be explained by assuming that L.acidophilus and L.helveticus, but not other species examined, use a single tRNA species for translation of family-box codons. Differences in expression level of genes are positively correlated with codon usage bias. Highly expressed genes show highly biased codon usage, whereas weakly expressed genes show much less biased codon usage. Codon usage patterns at the 5'-end of Lactobacillus genes is not significantly different from that of entire genes. The GC content of codons 2-6 is significantly reduced compared with that of the remainder of the gene. The possible implications of a reduced GC content for the control of translation efficiency are discussed.

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S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence

Cited by 129 publications

References 37 publications

Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA

Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA

Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Divergence in codon usage ofLactobacillus species

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