A large number of genes directly involved with translation and ribosomes exhibited increased expression following prolonged exposure to hyperosmotic stress. Ribosome structure integrity is significantly impaired by ionic strength of a medium (Brigotti et al., 2003), which in turn stalls translation. To contradict impaired translation L. monocytogenes appears to activate genes encoding numerous ribosomal proteins (Table 3.3).
Translation initiation factors encoding genes infC (lmo1785) and infA (lmo2610) both showed ≥ two- fold increase in transcription levels in three relatively salt tolerant L. monocytogenes strains (Table 3.3), whereas in strain 70-1700 infA showed no significant change in transcription. The translational elongation cycle is catalysed by three main elongation factors EF-Tu encoded by tufA (lmo2653), EF- Ts encoded by tsf (lmo1657) and EF-G encoded by fus (lmo2654), all of which showed up-regulation in the three relatively salt tolerant strains of L. monocytogenes following adaptation to hyperosmotic stress and only EF-Tu failed to show significant up-regulation in strain 70-1700 (Table 3.3).
Table 3. 3Gene expression patterns of four L. monocytogenes strains associated with translation and ribosome proteins observed during adaptive hyperosmotic stress response.
Gene ATCC19115 ScottA FW03-0035 70-1700 Function
LR# P LR P LR P LR P
cca 1.26 0.009 0.37 1.15 0.002 1.48 0.001 tRNA nucleotidyltransferase (CCA-adding enzyme)
efp 0.87 0.022 0.77 0.004 1.07 0.011 0.23 elongation factor EF-P
frr 2.00 0.000 1.07 0.011 1.45 1.4E-04 0.46 similar to ribosome recycling factor
fus 2.28 0.000 1.19 1.7E-04 1.42 3.1E-04 1.10 0.002 elongation factor EF-G
gatA 0.09 1.24 0.002 1.21 0.008 0.42 aspartyl/glutamyl-tRNA amidotransferase A
subunit
infA 1.20 0.017 1.19 0.006 1.21 0.002 0.18 translation initiation factor IF1
infC 1.30 0.019 1.21 0.003 0.78 0.61 translation initiation factor IF3
lmo0227 3.21 7.3E-05 2.68 4.1E-05 3.22 2.0E-06 1.32 0.003 putative tRNA-dihydrouridine lmo0935 1.53 0.002 0.81 0.003 1.10 0.001 1.00 0.001 putative rRNA methylase lmo1238 1.40 0.016 0.74 0.020 1.44 0.004 0.91 0.016 similar to ribonuclease PH lmo1530 0.68 1.19 0.001 1.42 0.005 1.01 0.005 similar to queuine tRNA-ribosyltransferase
97
Gene ATCC19115 ScottA FW03-0035 70-1700 Function
LR# P LR P LR P LR P
lmo1543 0.63 0.56 0.030 1.22 0.003 0.61 0.032 similar to ribonuclease G lmo1698 2.06 0.000 0.96 0.002 0.58 0.96 0.006 ribosomal-protein-alanine N-
acetyltransferase lmo1703 1.38 0.015 0.65 0.94 0.014 0.62 0.008 putative 23S rRNA (uracil-5-)-
methyltransferase lmo1709 1.60 0.019 0.65 0.019 0.37 0.54 0.014 similar to methionyl aminopeptidase lmo1722 1.04 1.09 0.002 0.91 0.013 0.70 0.013 putative ATP-dependent RNA helicase lmo1949 1.14 0.041 0.73 1.05 0.031 -0.32 similar to 23S RNA-specific pseudouridine
synthase B lmo2448 1.46 0.004 1.02 0.004 0.70 0.022 0.19 SsrA (tmRNA)-binding protein
rplA 3.37 0.001 1.90 7.7E-05 2.93 3.3E-05 1.08 0.002 ribosomal protein L1
rplB 0.91 0.81 0.019 1.26 0.003 0.16 ribosomal protein L2
rplC 1.69 0.002 0.82 0.012 1.40 0.002 1.06 0.015 ribosomal protein L3
rplD 2.08 0.000 1.31 3.8E-04 2.12 1.4E-06 0.94 3.6E-04 ribosomal protein L4
rplE 1.24 0.007 1.26 4.6E-04 2.03 4.2E-05 1.40 3.0E-05 ribosomal protein L5
rplF 1.22 0.009 1.05 0.016 1.90 3.4E-04 0.48 ribosomal protein L6
rplJ 2.50 1.8E-04 2.11 6.5E-05 2.99 4.4E-05 1.52 2.8E-04 ribosomal protein L10
rplK 3.36 0.001 1.49 4.8E-04 2.49 3.6E-04 1.79 1.5E-04 ribosomal protein L11
rplL 2.88 0.001 1.96 4.0E-04 2.58 3.2E-04 1.21 0.001 ribosomal protein L7/L12
rplM 0.81 1.54 0.001 1.44 0.018 0.73 ribosomal protein L13
rplN 1.96 0.001 1.45 0.001 3.18 8.8E-07 1.54 2.6E-04 ribosomal protein L14
rplR 1.90 0.001 1.34 0.002 2.37 1.9E-04 1.37 2.5E-05 ribosomal protein L18
rplS 1.94 0.001 1.29 2.2E-04 1.79 2.8E-05 0.78 0.017 ribosomal protein L19
rplT 1.29 0.006 0.90 0.007 0.84 0.005 0.48 ribosomal protein L20
rplU 2.74 4.31E-05 2.41 1.6E-04 2.28 3.5E-05 0.89 0.015 ribosomal protein L21
rplV 1.81 0.001 1.33 0.003 2.01 1.4E-04 0.80 0.001 ribosomal protein L22
rplW 2.05 0.002 0.85 1.53 0.005 0.32 ribosomal protein L23
rplX 1.13 0.014 1.03 0.013 1.58 0.001 0.41 ribosomal protein L24
rpmA 1.31 0.003 1.53 0.001 1.96 1.8E-05 0.91 0.021 ribosomal protein L27
rpmB 3.27 9.02E-05 3.01 0.001 3.52 8.1E-07 1.84 4.1E-04 ribosomal protein L28
rpmC 1.03 0.015 1.20 0.002 0.99 0.012 1.24 0.008 ribosomal protein L29
rpmD 2.44 4.2E-04 1.62 0.001 2.13 0.001 1.51 0.005 ribosomal protein L30
rpmE 2.59 4.3E-04 1.95 1.1E-04 2.89 4.7E-07 1.91 7.1E-05 ribosomal protein L31
rpmI 2.39 1.1E-04 1.80 3.3E-04 2.36 1.1E-06 1.38 9.0E-05 ribosomal protein L35
rpmJ 1.52 0.009 1.66 0.002 1.06 0.033 0.02 ribosomal protein L36
rpsB 2.22 3.6E-04 2.21 2.0E-04 1.45 0.002 0.30 ribosomal protein S2
rpsC 1.40 0.004 1.01 0.024 1.18 0.009 0.94 0.021 ribosomal protein S3
rpsD 3.21 0.001 1.03 0.001 2.34 0.001 1.48 4.7E-04 ribosomal protein S4
rpsE 1.11 0.006 0.84 0.003 1.39 3.9E-04 0.78 0.002 ribosomal protein S5
rpsG 3.41 2.6E-05 1.61 0.003 2.68 2.2E-07 0.79 0.048 ribosomal protein S7
rpsH 1.43 0.005 1.08 0.007 2.86 3.7E-06 1.69 1.1E-04 ribosomal protein S8
rpsJ 2.07 0.002 1.40 0.004 1.55 0.006 0.30 ribosomal protein S10
rpsK 1.36 0.003 0.67 1.19 1.2E-04 0.55 0.025 ribosomal protein S11
rpsL 4.10 5.9E-06 1.81 4.6E-04 2.59 3.6E-08 1.43 2.0E-04 ribosomal protein S12
rpsM 1.73 0.001 1.44 0.004 1.27 2.7E-04 0.32 ribosomal protein S13
rpsN 1.68 0.001 1.74 0.001 1.80 3.6E-05 0.71 0.004 ribosomal protein S14
98
Gene ATCC19115 ScottA FW03-0035 70-1700 Function
LR# P LR P LR P LR P
rpsS 2.24 0.001 1.30 0.001 2.21 2.0E-05 1.18 9.9E-05 ribosomal protein S19
rpsT 1.23 0.007 0.33 0.81 1.66 0.024 ribosomal protein S20
tsf 1.46 0.001 1.28 4.7E-05 1.52 4.1E-04 1.03 2.6E-04 elongation factor EF-Ts
tufA 2.02 0.000 1.29 0.002 1.58 2.6E-03 0.79 elongation factor EF-Tu
# LR= Log Ratio; P values not shown were >0.05
EF-Tu is a small GTP-binding protein and in combination with a GTP molecule is responsible for transporting and binding of the appropriate codon-specified aminoacyl-tRNA to the acceptor site on the ribosome. EF-Ts is a guanine nucleotide exchange factor acting on EF-Tu, allowing its reactivation by mediating GDP/GTP exchange. In addition to its role in translation and elongation EF-Tu has been shown to have protective influence on newly synthesized proteins in terms of protein folding and renaturation after stress (Caldas et al., 1998; Duché et al., 2002). EF-G is a small GTP-binding protein, which is required for translocation of ribosomes on the mRNA molecule. ATCC19115, ScottA and FW03-0035 also showed increase in transcription of efp (lmo1355) a gene encoding the less well known elongation factor EF-P (Table 3.3). EF-P is believed to be involved in a formation of a first peptide bond of a protein molecule (Ganoza et al., 2002; Hanawa-Suetsugu et al., 2004). The gene encoding ribosomal recycling factor, frr (lmo1314) was up-regulated in ATCC19115, ScottA and FW03-0035 strains and showed no significant change in transcription in 70-1700 (Table 3.3). Ribosome recycling factor in conjunction with EF-G is required for dissociation of post-termination ribosomal complex after release of a newly synthesized polypeptide (Pai et al., 2008; Toyoda et al., 2000).
Aminoacyl-tRNA synthetases are key components of the protein translation machinery that catalyse activation of amino acids via the formation of aminoacyl adenylatesand linking the activated amino acid to the cognate tRNAs (Hausmann & Ibba, 2008; Wolf et al., 1999). Genes in this subgroup showed significant up-regulation in all four strains of L. monocytogenes with T-value scores ranging from 2.04 to 3.50 (Table 3.1). Activation of this group of genes has also been observed in B. sublilis
following adaptation to osmotic stress (Hahne et al., 2010).Four genes in this group showed up- regulation in all four strains of L. monocytogenes, these were hisS (lmo1520) encoding histidyl-tRNA synthetase, trpS (lmo2198) encoding tryptophanyl-tRNA synthetase, proS (lmo1319) encoding prolyl- tRNA synthetase and aspS (lmo1519) encoding aspartyl-tRNA synthetase (Fig. 3.19).
99
*=significance level of Log Ratio for each gene * p<0.05; ** p<0.01,*** p<0.001, ns p>0.05
Figure 3.19Expression of genes associated with aminoacyl-tRNA biosynthesis in four osmo-adapted L. monocytogenes strains.
Increased transcription in cells adapted to hyperosmotic stress is evident from the observed gene expression profiles and is suggestive of a requirement of these cells for amplified protein synthesis for utilization in cell-modifications to assist in repair of NaCl induced cell damage.
The expression of RNA polymerase delta (δ) factor (lmo2560) has shown on average 2-fold increased in all four strains following adaptation to hyperosmotic stress (Fig. 3.18). The exact role of bacterial
δ-factor is still very poorly understood, although it has been shown to have a global affect on gene expression in B. subtilis, where it is thought to participate in maintaining transcriptional specificity and RNA polymerase recycling (Lopez de Saro et al., 1999; Seepersaud et al., 2006). A mutation in δ
has also been reported to affect the ability of human pathogen S. aureus to recover from nutrient starvation and increase its sensitivity to acid stress (Watson et al., 1998). Association of δ-factor with RNA polymerase reduces binding to DNA templates containing non relevant promoter sites, which may be of benefit in an organism straggling to maintain normal physiological function when exposed to environmental stress such as hyperosmotic stress. Prevention of non-specific RNA polymerase binding to DNA may reduce energy wastage by increasing the amount of free RNA polymerase within the bacterial cell.
Defective messages are frequently produced during growth and can be critical for cell survival especially when bacterium is under stress. A number of mechanisms exist in bacteria which control the quality of the message pre- and post-translation, the trans-translation reaction involving
transfer-messenger RNA (tmRNA) is one such mechanism. The trans-translation reaction directs
0 1 2 aspS proS argS trpS hisS lysS metS
ATCC19115
** ** * * Log Ratio * * * 0 1 2ScottA
** *** ** * * *** ** 0 1 2FW03-0035
* ** *** *** ns *** * 0 1 270-1700
ns ns * ns *** *** *100 incompletely synthesized peptides to degradation and releases stalled ribosomes and assists in the adaptation to stress (Fujihara et al., 2002; Gillet & Felden, 2001; Withey & Friedman, 2003). In both
E. coli and B. subtilis, tmRNA has been shown to assist bacterial cells in adaptation to environmental change, including elevated temperatures and to exposure to ethanol and cadmium chloride (Fujihara
et al., 2002; Gillet & Felden, 2001; Withey & Friedman, 2003). The ssrA gene (lmo2448) to date has not been linked to stress adaptation in L. monocytogenes. Marginal increase in the level of tmRNA conscript was observed in three out of four strains of L. monocytogenes following exposure to prolonged hyperosmotic stress in this study (Table 3.3). It appears that the quality control mediated by tmRNA might be of benefit in ensuring that the proteins allowing growth under hyperosmotic stress are produced and the consequences of translational errors that are expected to increase are corrected.